Polymer-silica hybrid pdots and methods of use thereof

ABSTRACT

The present disclosure provides organic-inorganic hybrid polymer particles, which have desirable surface chemistry and optical properties that make them particularly suitable for biological and optical applications. The present disclosure also provides methods of making organic-inorganic hybrid polymer particles. The present disclosure also provides methods of using the organic-inorganic hybrid polymer particles for biological and optical applications.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a Continuation of co-pending U.S. patent applicationSer. No. 16/900,809, filed Jun. 12, 2019, which is a Continuation ofU.S. patent application Ser. No. 16/309,795, filed Dec. 13, 2018, nowU.S. Pat. No. 10,770,197, issued Sep. 8, 2020, which is a U.S. NationalPhase Application under 35 U.S.C. § 371 of International PatentApplication No. PCT/US2017/037260, filed Jun. 13, 2017, now expired,which claims the benefit of U.S. Provisional Patent Application No.62/350,126, filed Jun. 14, 2016, now expired, the disclosures of whichare incorporated herein by reference in their entireties.

BACKGROUND

Advances in understanding biological systems have relied on applicationsof fluorescence microscopy, flow cytometry, versatile biological assays,and biosensors. These experimental approaches make extensive use oforganic dye molecules as probes. But intrinsic limitations of theseconventional dyes such as low absorptivity, and poor photostability haveposed great difficulties in further developments of high-sensitivityimaging techniques and high-throughput assays. As a result, there hasbeen considerable interest in developing brighter and more photostablefluorescent nanoparticles.

Traditional chromophoric polymer dots have been studied for imaging anddetection techniques for researching chemical and biological analytesand systems. Functionalization of chromophoric polymer dots for use inbioconjugation has been attempted, but problems with polymer dotswelling, instability, and aggregation in biological buffer solutions,as well as nonspecific interactions in certain environments have beenencountered.

SUMMARY

The present disclosure provides a new class of organic-inorganic hybridpolymer dots and related methods.

In various aspects, the present disclosure provides an organic-inorganichybrid polymer dot comprising: a semiconducting chromophoric polymer;and an inorganic network, wherein the semiconducting chromophoricpolymer and the inorganic network form an organic-inorganicinterpenetrated network.

In various aspects, the present disclosure provides a method of makingan organic-inorganic hybrid polymer dot, the method comprising:providing a solution, wherein the solution comprises a solvent, asemiconducting chromophoric polymer, and an organo-silane; and mixingthe solution with an aqueous solution, wherein at least one of thesolution or the aqueous solution comprises an organo-silane comprisingX, wherein X is a functional group suitable for bioconjugation.Preferably or optionally, the solution can also comprise an additionalsilane that can help to make the hybrid polymer dot smaller and/or morecompact.

In various aspects, the present disclosure provides an organic-inorganicinterpenetrated hybrid chromophoric polymer dot comprising asemiconducting chromophoric polymer, an inorganic network, and afunctional group that is suitable for bioconjugation.

In various aspects, the present disclosure provides an organic-inorganichybrid polymer dot comprising: a semiconducting chromophoric polymer; X,wherein X is a functional group suitable for bioconjugation; and aninorganic network that is covalently bonded to the semiconductingchromophoric polymer.

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 provides a schematic illustration of a method for preparinghybrid polymer dots.

FIG. 2 illustrates a method of preparing carboxylate functionalized PFBThybrid polymer dots.

FIG. 3 illustrates Transmission Electron Microscopy (TEM) images of thehybrid polymer dots.

FIG. 4 illustrates comparative single particle fluorescence curves ofbare PFBT polymer dots and the hybrid polymer dots prepared from PFBT,alkylsilane, and TEOS at different ratios.

FIG. 5 illustrates cellular labeling brightness for different hybridpolymer dot bioconjugates as well as bare polymer dot bioconjugates, asquantified by flow cytometry.

FIG. 6 shows fluorescence imaging of MCF cells specifically labeled withthe hybrid polymer dot bioconjugates based on the blending set ofPFBT/TMOS/TEOS.

FIG. 7 illustrates photostability of the MCF cells labeled with thehybrid polymer dot bioconjugates.

FIG. 8 shows fluorescence imaging of MCF cells specifically labeled withthe hybrid polymer dot bioconjugates based on the blending set ofPFBT/TCOS/TEOS.

FIG. 9 illustrates photostability curves of the MCF cells labeled withthe hybrid polymer dot bioconjugates.

FIG. 10 illustrates results of gel electrophoresis of the hybrid polymerdots and related bioconjugates.

FIG. 11 shows flow cytometry results of MCF-7 cells labeled with thehybrid polymer dots.

FIG. 12 provides a general schematic illustration of conjugated polymerswith a silane chain and functional silane chain for bioconjugation.

FIG. 13 provides a general schematic illustration of conjugated polymerswith a silane chain and functional chain for bioconjugation.

FIG. 14 provides a general schematic illustration of conjugated polymerswith a functional silane chain for bioconjugation.

FIG. 15 illustrates a method of preparing carboxylate functionalizedPFBT hybrid polymer dots with the use of a pre-functionalized PFBTpolymer.

FIG. 16 shows flow cytometry results of MCF-7 cells labeled withfunctionalized PFBT hybrid dots with 14% of monomeric units comprisingC₂COOH (i.e., PFBT-14% C₂COOH), in comparison to those labeled with theSilane-COONa hybrid polymer dots.

FIG. 17 provides a TEM image of the PFBT-14% C₂COOH hybrid polymer dots.

DETAILED DESCRIPTION

The present disclosure provides compositions of, as well as relatedmethods of making and using, organic-inorganic hybrid polymer dots,which have desirable surface chemistry and optical properties that makethem particularly suitable for biological applications. These and otherembodiments are described in detail herein.

The invention will best be understood by reference to the followingdetailed description of the embodiments and embodiments of theinvention, taken in conjunction with the accompanying drawings andfigures. The discussion below is descriptive, illustrative, andexemplary and is not to be taken as limiting the scope defined by anyappended claims.

Various polymer compositions are suitable for use with the embodimentsherein. In some embodiments, a “polymer” is a molecule composed of atleast two repeating structural units typically connected by covalentchemical bonds. A polymer of the present disclosure can have differentkinds of repeating units, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10different kinds of repeating units. The repeating structural unit may beone type of monomer, and the resulting polymer is a homopolymer. In someembodiments, the polymers can include two different types of monomers,three different types of monomers, four different types of monomers,five different types of monomers, or more types of monomers. One ofordinary skill in the art will appreciate that the different types ofmonomers can be distributed along a polymer chain in a variety of ways.For example, three different types of monomers can be randomlydistributed along the polymer. It will similarly be appreciated that thedistribution of monomers along the polymer can be represented indifferent ways. The number of repeating structural units (e.g.,monomers) along the length of a polymer can be represented by “n.” Insome embodiments, n can range, e.g., from at least 2, from at least 10,from at least 50, from at least 100, from at least 500, from at least1,000, from at least 10,000, or higher. In certain embodiments, n canrange from 2 to 10,000, from 10 to 10,000, from 10 to 1,000, from 20 to5,000, from 20 to 500, from 50 to 300, from 100 to 1,000, from 100 to10,000, or from 500 to 10,000.

In some embodiments, polymers have extended molecular structurescomprising backbones that optionally contain pendant side groups. Thepolymers provided herein can include, but are not limited to, linearpolymers and branched polymers such as star polymers, comb polymers,brush polymers, ladders, and dendrimers. As described further herein,the polymers can include semiconducting polymers generally well known inthe art.

In some embodiments, a “polymer particle,” “polymeric particle,” or“Pdot” is a sub-micrometer-sized entity, which represents a separatediscontinuous phase surrounded by a continuous free-flowing medium. Thefree flowing medium is usually a low-molecular-weight liquid, most oftenwater. In some embodiments, the terms “polymer particle,” “polymericparticle,” or “Pdot” can be used interchangeably.

In some embodiments, the terms “polymer particle,” “hybrid polymer dot,”“polymer dot,” “chromophoric polymer dot,” “chromophoric semiconductingpolymer dot,” “fluorescent polymer dot,” “chromophoric nanoparticle” and“Pdot” are used interchangeably to refer to structures comprising one ormore polymers (e.g., semiconducting polymers, non-semiconductingpolymers, or a combination thereof) that have been collapsed into astable sub-micron-sized particle. Various methods are suitable forforming hybrid polymer dots, as described further herein. The hybridpolymer dots provided herein can be made up of a single polymer or cancomprise blends of polymers. In certain embodiments, the one or morepolymers are collapsed, precipitated, and/or condensed to form a polymermatrix. In some embodiments, the properties of the hybrid polymer dotsare dependent on the polymer structures. Therefore, the polymer backbone(main chain), side chains, terminal units, and substituted groups can bevaried to obtain specific properties. In some embodiments, the opticalproperties of the hybrid polymer dots can be tuned by varying thestructures of the polymer backbone (main chain).

In certain embodiments, the hybrid polymer dots provided herein includeone or more chromophores, also referred to herein as chromophoric units.In some embodiments, the term “chromophore” or “chromophoric unit” isgiven its ordinary meaning in the art. A chromophore absorbs certainwavelengths of light, e.g., from the UV region to the near infraredregion, and may be or may not be emissive. In some embodiments, achromophoric unit includes, but is not limited to, a unit of structureswith delocalized pi-electrons, a unit of small organic dye molecules,and/or a unit of metal complexes. The chromophore can be part of thepolymer matrix or be incorporated into the polymer matrix, e.g., byblending, crosslinking, and the like.

In certain embodiments, the hybrid polymer dots of the presentdisclosure include one or more chromophoric polymers. In someembodiments, the term “chromophoric polymer” refers to a polymer inwhich at least a portion of the polymer absorbs certain wavelengths oflight, e.g., ranging from UV to near infrared spectra. Chromophoricpolymers according to the present disclosure may be or may not beemissive. In some embodiments, a “chromophoric polymer” is a polymer inwhich at least a portion of the polymer includes chromophoric units.Examples of chromophoric polymers can include polymers comprising unitsof structures with delocalized pi-electrons such as semiconductingpolymers, polymers comprising units of small organic dye molecules,polymers comprising units of metal complexes, and polymers comprisingunits of any combinations thereof. The chromophoric unit can beincorporated into the polymer backbone. The chromophoric unit can alsobe covalently attached to the side chain, or the terminal unit of thepolymer. Chromophoric polymers can be made using standard synthesismethods generally well known in the art.

In certain embodiments, the chromophoric polymer is a “conjugatedpolymer.” The term “conjugated polymer” is recognized in the art.Electrons, holes, or electronic energy, can be conducted along theconjugated structure. In some embodiments, a large portion of thepolymer backbone can be conjugated. In some embodiments, the entirepolymer backbone can be conjugated. In some embodiments, the polymer caninclude conjugated structures in their side chains or termini. In someembodiments, the conjugated polymer can have conducting properties,e.g., the polymer can conduct electricity. In some embodiments, theconjugated polymer can have semiconducting properties, e.g., thepolymers can exhibit a direct band gap, leading to an efficientabsorption or emission at the band edge. Therefore, in certainembodiments, the chromophoric polymer is a “semiconducting polymer.” Theterm “semiconducting polymer” is recognized in the art.

In some embodiments, the term “alkyl” refers to a straight or branched,saturated, aliphatic radical having the number of carbon atomsindicated. For example, C₁-C₆ alkyl includes, but is not limited to,methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include,but are not limited to heptyl, octyl, nonyl, decyl, etc. Alkyl caninclude any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7,1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. Thealkyl group is typically monovalent, but can be divalent, such as whenthe alkyl group links two moieties together. As used herein, the term“heteroalkyl” refers to a straight or branched, saturated, aliphaticradical of carbon atoms, where at least one of the carbon atoms isreplaced with a heteroatom, such as N, O or S. Additional heteroatomscan also be useful, including, but not limited to, B, Al, Si and P.

In some embodiments, the term “alkylene” refers to an alkyl group, asdefined above, linking at least two other groups, i.e., a divalenthydrocarbon radical. The two moieties linked to the alkylene can belinked to the same atom or different atoms of the alkylene. Forinstance, a straight chain alkylene can be the bivalent radical of—(CH₂)_(n), where n is 1, 2, 3, 4, 5 or 6. Alkylene groups include, butare not limited to, methylene, ethylene, propylene, isopropylene,butylene, isobutylene, sec-butylene, pentylene and hexylene.

In some embodiments, the term “alkoxy” refers to an alkyl group havingan oxygen atom that either connects the alkoxy group to the point ofattachment or is linked to two carbons of the alkoxy group. Alkoxygroups include, for example, methoxy, ethoxy, propoxy, iso-propoxy,butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy,etc. The alkoxy groups can be further substituted with a variety ofsubstituents described within. For example, the alkoxy groups can besubstituted with halogens to form a “halo-alkoxy” group.

In some embodiments, the term “alkenyl” refers to either a straightchain or branched hydrocarbon of 2 to 6 carbon atoms, having at leastone double bond. Examples of alkenyl groups include, but are not limitedto, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl,butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl,1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl,1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl.

In some embodiments, the term “alkynyl” refers to either a straightchain or branched hydrocarbon of 2 to 6 carbon atoms, having at leastone triple bond. Examples of alkynyl groups include, but are not limitedto, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl,butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl,1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl,1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl.

As used herein, the term “alkynylene” refers to an alkynyl group, asdefined above, linking at least two other groups, i.e., a divalenthydrocarbon radical. The two moieties linked to the alkynylene can belinked to the same atom or different atoms of the alkynylene. Alkynylenegroups include, but are not limited to, ethynylene, propynylene,isopropynylene, butynylene, sec butynylene, pentynylene and hexynylene.

In some embodiments, the term “alkyl amine” refers to an alkyl group asdefined within, having one or more amino groups. The amino groups can beprimary, secondary or tertiary. The alkyl amine can be furthersubstituted with a hydroxy group. Alkyl amines can include, but are notlimited to, ethyl amine, propyl amine, isopropyl amine, ethylene diamineand ethanolamine. The amino group can link the alkyl amine to the pointof attachment with the rest of the compound, be at the omega position ofthe alkyl group, or link together at least two carbon atoms of the alkylgroup.

In some embodiments, the term “halogen” or “halide” refers to fluorine,chlorine, bromine and iodine. As used herein, the term “haloalkyl”refers to alkyl as defined above where some or all of the hydrogen atomsare substituted with halogen atoms. Halogen (halo) preferably representschloro or fluoro, but may also be bromo or iodo. As used herein, theterm “halo-alkoxy” refers to an alkoxy group having at least onehalogen. Halo-alkoxy is as defined for alkoxy where some or all of thehydrogen atoms are substituted with halogen atoms. The alkoxy groups canbe substituted with 1, 2, 3, or more halogens. When all the hydrogensare replaced with a halogen, for example by fluorine, the compounds areper-substituted, for example, perfluorinated. Halo-alkoxy includes, butis not limited to, trifluoromethoxy, 2,2,2,-trifluoroethoxy,perfluoroethoxy, and the like.

In some embodiments, the term “cycloalkyl” refers to a saturated orpartially unsaturated, monocyclic, fused bicyclic or bridged polycyclicring assembly containing from 3 to 12 ring atoms, or the number of atomsindicated. Monocyclic rings include, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic andpolycyclic rings include, for example, norbomane, decahydronaphthaleneand adamantane. For example, C₃₋₈cycloalkyl includes cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbomane.

In some embodiments, the term “cycloalkylene” refers to a cycloalkylgroup, as defined above, linking at least two other groups, i.e., adivalent hydrocarbon radical. The two moieties linked to thecycloalkylene can be linked to the same atom or different atoms of thecycloalkylene. Cycloalkylene groups include, but are not limited to,cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, andcyclooctylene.

In some embodiments, the term “heterocycloalkyl” refers to a ring systemhaving from 3 ring members to about 20 ring members and from 1 to about5 heteroatoms such as N, O and S. Additional heteroatoms can also beuseful, including, but not limited to, B, Al, Si and P. The heteroatomscan also be oxidized, such as, but not limited to, —S(O)— and —S(O)₂—.

In some embodiments, the term “heterocycloalkylene” refers to aheterocycloalkyl group, as defined above, linking at least two othergroups. The two moieties linked to the heterocycloalkylene can be linkedto the same atom or different atoms of the heterocycloalkylene.

In some embodiments, the term “aryl” refers to a monocyclic or fusedbicyclic, tricyclic or greater, aromatic ring assembly containing 6 to16 ring carbon atoms. For example, aryl may be phenyl, benzyl, azulenylor naphthyl. Aryl groups can be mono-, di- or tri-substituted by one,two or three radicals selected from alkyl, alkoxy, aryl, hydroxy,halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy andoxy-C₂-C₃-alkylene; all of which are optionally further substituted, forinstance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or2-phenanthrenyl. Alkylenedioxy is a divalent substitute attached to twoadjacent carbon atoms of phenyl, e.g., methylenedioxy or ethylenedioxy.Oxy-C₂-C₃-alkylene is also a divalent substituent attached to twoadjacent carbon atoms of phenyl, e.g., oxyethylene or oxypropylene. Anexample for oxy-C₂-C₃-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

Aryl groups can include, but are not limited to, naphthyl, phenyl orphenyl mono- or disubstituted by alkoxy, phenyl, halogen, alkyl ortrifluoromethyl, phenyl or phenyl-mono- or disubstituted by alkoxy,halogen or trifluoromethyl, and in particular phenyl.

In some embodiments, the terms “alkoxy-aryl” refers to an aryl group, asdefined above, where one of the moieties linked to the aryl is linkedthrough an oxygen atom. Alkoxy-aryl groups include, but are not limitedto, phenoxy (C₆H₅O—). The present disclosure also includesalkoxy-heteroaryl groups.

In some embodiments, the term “heteroaryl” refers to a monocyclic orfused bicyclic or tricyclic aromatic ring assembly containing 5 to 16ring atoms, where from 1 to 4 of the ring atoms are a heteroatom each N,O or S. For example, heteroaryl includes pyridyl, indolyl, indazolyl,quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl,furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl,triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any otherradicals substituted, especially mono- or di-substituted, by e.g.,alkyl, nitro or halogen. Similarly, aryl and heteroaryl groups describedherein can be substituted or unsubstituted. Substituents for the aryland heteroaryl groups are varied, such as alkyl, aryl, CN, amino,sulfide, aldehyde, ester, ether, carboxyl, hydroxyl or halide.Substituents can be a reactive group, such as but not limited to chloro,bromo, iodo, hydroxyl, or amino. Substituents can be selected from:halogen, OR′, OC(O)R′, NR′R″, SR′, R′, CN, NO₂, CO₂R′, CONR′R″, C(O)R′,OC(O)NR′R″, NR″C(O)R′, NR″C(O)₂R′, NR′C(O)NR″R′″, NHC(NH₂)═NH,NR′C(NH₂)═NH, NH C(NH₂)═NR′, S(O)R′, S(O)₂R′, S(O)₂NR′R″, NS, CH(Ph)₂,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″ and R′″ are independentlyselected from hydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryland heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl.

The groups described herein can be substituted or unsubstituted.Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups, such as alkyl, aryl,cyano (CN), amino, sulfide, aldehyde, ester, ether, carboxyl, hydroxylor halide. Substituents can be a reactive group, such as but not limitedto chloro, bromo, iodo, hydroxyl, or amino. Suitable substituents can beselected from: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NH—C(NH₂)═NH, —NR′C(NH₂)═NH,—NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂ in a numberranging from zero to (2m′+1), where m′ is the total number of carbonatoms in such radical. R′, R″ and R′″ each independently refer tohydrogen, unsubstituted (C₁-C₈)alkyl and heteroalkyl, unsubstitutedaryl, alkoxy or thioalkoxy groups, or aryl-(C₁-C₄)alkyl groups. When R′and R″ are attached to the same nitrogen atom, they can be combined withthe nitrogen atom to form a 5-, 6-, or 7-membered ring. For example,—NR′R″ is meant to include 1-pyrrolidinyl and 4-morpholinyl. From theabove discussion of substituents, one of skill in the art willunderstand that the term “alkyl” is meant to include groups such ashaloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃,—C(O)CH₂OCH₃, and the like).

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a compound”includes a plurality of such compounds, reference to “an agent” includesa plurality of such agents, and reference to “the cell” includesreference to one or more cells (or to a plurality of cells) andequivalents thereof known to those skilled in the art, and so forth.When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and subcombinations of ranges and specific embodimentstherein are intended to be included. The term “about” when referring toa number or a numerical range means that the number or numerical rangereferred to is an approximation within experimental variability (orwithin statistical experimental error), and thus the number or numericalrange may vary between 1% and 15% of the stated number or numericalrange. The term “comprising” (and related terms such as “comprise” or“comprises” or “having” or “including”) is not intended to exclude thatin other certain embodiments, for example, an embodiment of anycomposition of matter, composition, method, or process, or the like,described herein, may “consist of” or “consist essentially of” thedescribed features.

As used herein the term “and/or” is used as a functional word toindicate that two words or expressions are to be taken together orindividually. For example, A and/or B encompasses A alone, B alone, andA and B together.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the disclosure provided herein. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and are also encompassed within thedisclosure, subject to any specifically excluded limit in the statedrange. Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe disclosure provided herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the disclosure, the preferredmethods, devices and materials are now described.

Organic-Inorganic Hybrid Polymer Dots

The present disclosure provides various embodiments of organic-inorganichybrid polymer dots, also referred to herein as “hybrid polymer dots.”In some embodiments, an organic-inorganic hybrid polymer dot comprisesan organic network and an inorganic network. In certain embodiments, theorganic network includes at least one organic species, such as one ormore of the chromophoric polymers described herein. In certainembodiments, an inorganic network comprises at least one inorganicspecies, such as siloxane, alumino-siloxane, titanium-siloxane, titaniumoxide, or a combination thereof. In certain embodiments, an inorganicnetwork is a siloxane network (e.g., including Si—O—Si linkages), analumino-siloxane network (e.g., including Al—O—Si linkages), atitanium-siloxane network (e.g., including Ti—O—Si linkages), a titaniumoxide network (e.g., including Ti—O—Ti linkages), or a combinationthereof. Additional examples of inorganic networks such as siloxanenetworks are discussed in further detail herein. The terms “siloxanenetwork” and “silica (SiO₂) network” treated synonymously herein.

In some embodiments, the organic network and inorganic network areinterpenetrated with each other so as to form an organic-inorganicinterpenetrated network. For example, a siloxane network can form aninterpenetrated network with a chromophoric polymer. As used herein, an“organic-inorganic interpenetrated network” refers to the polymer dotmatrix comprising at least two networks that together form theinterpenetrated network. In some embodiments, the organic-inorganicinterpenetrated network is mesh-like and/or an interlocking structure ofthe inorganic network interpenetrated with the polymer. In someembodiments, interpenetration occurs primarily through the physicalassociation (e.g., hydrophobic interaction) of the at least two networksso as to form the interpenetrated network. In certain embodiments,interpenetration occurs through the physical association of the at leasttwo networks without any chemical bonding (e.g., without covalentbonding between the two networks). In certain embodiments,interpenetration occurs primarily through the chemical bonding (e.g.,covalent bonding) of the two networks to each other so as to form theinterpenetrated network. Covalent bonding between the organic networkand inorganic network can be used alternatively to or in combinationwith physical association in order to form the organic-inorganicinterpenetrated network.

In certain embodiments, the present disclosure providesorganic-inorganic hybrid polymer dots that are structurally distinctfrom other types of polymer dots and particles, included but not limitedto polymer dots formed by blending (e.g., polymer dots blended withamphiphilic polymers) and polymer dots without an inorganic network. Forexample, in some embodiments, the organic-inorganic interpenetratednetwork of the hybrid polymer dots described herein is distinct from acore-cap or core-shell structure that may be found in other types ofpolymer dots. In certain embodiments, the organic-inorganic hybridpolymer dots herein do not include a core-cap or core-shell structure.

As described in further detail herein, in some embodiments, theorganic-inorganic interpenetrated network is formed during formation ofthe organic-inorganic hybrid polymer dot. For example, in someembodiments, formation of an organic-inorganic hybrid polymer dotinvolves forming a siloxane network during hydrolysis of organic silanemolecules. In certain embodiments, the organic silane is an alkylsilane.In certain embodiments, one or more polymers are collapsed,precipitated, or condensed simultaneously with hydrolysis of organicsilane molecules and cross linking in order to simultaneously form anorganic network and an inorganic network, which together form theorganic-inorganic interpenetrated network.

The weight percent of the inorganic network (e.g., siloxane network)and/or the components thereof (e.g., silicon (Si)) in a hybrid polymerdot can be varied as desired. In some embodiments, the weight percent ofthe inorganic network (e.g., siloxane network) and/or the componentsthereof (e.g., silicon) is selected to avoid formation of a core-shellstructure in the resulting hybrid polymer dot. In certain embodiments,the weight percent of silicon from the inorganic network in the hybridpolymer dot is less than or equal to about 1%, less than or equal toabout 5%, less than or equal to about 10%, less than or equal to about15%, less than or equal to about 20%, less than or equal to about 25%,less than or equal to about 30%, less than or equal to about 35%, lessthan or equal to about 40%, less than or equal to about 45%, or lessthan or equal to about 47%. In certain embodiments, the weight percentof silicon from the inorganic network in the hybrid polymer dot isgreater than or equal to about 1%, greater than or equal to about 5%,greater than or equal to about 10%, greater than or equal to about 15%,greater than or equal to about 20%, greater than or equal to about 25%,greater than or equal to about 30%, greater than or equal to about 35%,greater than or equal to about 40%, or greater than or equal to about45%. In certain embodiments, the weight percent of silicon from theinorganic network in the hybrid polymer dot is within a range from about1% to about 45%, or within a range from about 1% to about 47%.

The hybrid polymer dots of the present disclosure can be functionalizedand/or bioconjugated, e.g., to a biological molecule. In someembodiments, a hybrid polymer dot includes an organic network (e.g., asemiconducting chromophoric polymer), an inorganic network (e.g., asiloxane network), and X, where X is a functional group suitable forbioconjugation. Examples of functional groups and/or linkers suitablefor bioconjugation in accordance with the present disclosure areprovided further below. The functional group X may be attached to theinorganic network, the organic network, or a combination thereof. Incertain embodiments the functional group is attached to the inorganicnetwork. In certain embodiments the functional group is attached to thesemiconducting chromophoric polymer. In certain embodiments thefunctional group comprises a hydrophobic functional group, a hydrophilicfunctional group, or a combination thereof. In certain embodiments thefunctional group is suitable for bioconjugation.

In some embodiments, a hybrid polymer dot includes at least twoorthogonal reactive chemical groups. In certain embodiments, anorthogonally reactive chemical group is a chemical group that reactsonly with its designated chemical reactive group, but not with anotherchemical reactive group that may be present. For example, reactivegroups A and B can form a designated pair that reacts with each other,and reactive groups Y and Z can form another designated pair that reactswith each other. In such embodiments, reactive group A is considered tobe orthogonal with respect to Y because A does not react with Z, andreactive group Y is orthogonal with respect to A because Y does notreact with B. In some embodiments, reactive groups A can react with eachother or with reactive groups B to form a siloxane network, and reactivegroups Y do not react with either A or B, such that A and Y, and/or Band Y, are considered to be orthogonal reactive groups.

In some embodiments, the hybrid polymer dot includes a semiconductingchromophoric polymer that includes at least two orthogonal reactivechemical groups. In certain embodiments, at least one of the orthogonalreactive chemical groups has the formula C_(n)H_(2n)X or C_(n)F_(2n)X,wherein X is a functional group suitable for bioconjugation as describedfurther herein and n is not less than 1.

In some embodiments, organic-inorganic hybrid polymer dots comprise atleast two inorganic species, each having its own respective function.For example, the organic-inorganic hybrid polymer dots can have asurface that is functionalized with a functional species comprising acarboxyl, an amine, a thiol (—SH), a carboxylate or carboxylic acid, amaleimide, a maleic anhydride, a N-hydroxysuccinimide (NHS), an alcohol(—OH), or a cyanate, or a combination thereof that is suitable forbioconjugation, as discussed further herein. Additionally, hybridpolymer dots can comprise an aliphatic chain, for example, an alkylchain. The aliphatic chain can take part in cross-linking duringformation of the interpenetrated network. The aliphatic chain can bephysically associated with the organic network (e.g., chromophoricpolymer) and/or the inorganic network during formation of theinterpenetrated network.

The resulting hybrid polymer dots display a set of advantageousproperties for biological applications. For example, theorganic-inorganic interpenetrated network of the hybrid polymer dotsallows for robust, compact polymer dots with high fluorescencebrightness by, for example, preventing undesirable chain-chaininteractions. For example, the interpenetrated network of the hybridpolymer dots may decrease undesirable fluorescent quenching. Theorganic-inorganic hybrid polymer dots exhibit high fluorescence quantumyield, improved photostability, and improved colloidal stability.Therefore, the fluorescence quantum yield and photostability of thehybrid polymer dots can be significantly improved. The hybrid polymerdots are stable in a range of biological media and do not swell or formaggregates in a variety of biological buffers.

As used herein, the term “stable,” in reference to polymer dots, canrefer to polymer dots that do not aggregate and/or change substantiallyin size (as measured by electron microscopy, atomic force microscopy, ordynamic light scattering) when stored in an appropriate aqueous solutionfor an extended period of time. Aggregation or a change substantially insize of the polymer dots can, for example, be characterized as anincreasing number of aggregates including more than one polymer dot.Aggregates can be detected visually by the eye, with imaging techniques,such as electron microscopy or atomic for microscopy, and/or byincreased size measurements shown by dynamic light scattering.

In some embodiments, hybrid polymer dots can have a diameter of not lessthan 5 nm and not greater than 1,000 nm. In some embodiments, theorganic-inorganic hybrid polymer dots disclosed herein can have adiameter of not less than 5 nm and not greater than 500 nm. In someembodiments, the organic-inorganic hybrid polymer dots disclosed hereincan have a diameter of not less than 5 nm and not greater than 100 nm.In some embodiments, the organic-inorganic hybrid polymer dots disclosedherein can have a diameter of not less than 5 nm and not greater than 50nm. In some embodiments, the organic-inorganic hybrid polymer dotsdisclosed herein can have a diameter of not less than 10 nm and notgreater than 30 nm. In some embodiments, the organic-inorganic hybridpolymer dots disclosed herein can have a diameter of not greater than100 nm. In some embodiments, the organic-inorganic hybrid polymer dotsdisclosed herein can have a diameter not greater than 50 nm. In someembodiments, the organic-inorganic hybrid polymer dots disclosed hereincan have a diameter of not greater than 40 nm. In some embodiments, theorganic-inorganic hybrid polymer dots disclosed herein can have adiameter of not greater than 30 nm. In some embodiments, theorganic-inorganic hybrid polymer dots disclosed herein can have adiameter of not greater than 20 nm. In some embodiments, theorganic-inorganic hybrid polymer dots disclosed herein can have adiameter of not less than 5 nm and not greater than 300 nm. In someembodiments, the organic-inorganic hybrid polymer dots disclosed hereincan have a diameter of not less than 5 nm and not greater than 200 nm.In some embodiments, the organic-inorganic hybrid polymer dots disclosedherein can have a diameter of not less than 5 nm and not greater than150 nm. In some embodiments, the organic-inorganic hybrid polymer dotsdisclosed herein can have a diameter of not less than 5 nm and notgreater than 90 nm. In some embodiments, the organic-inorganic hybridpolymer dots disclosed herein can have a diameter of not less than 5 nmand not greater than 80 nm. In some embodiments, the organic-inorganichybrid polymer dots disclosed herein can have a diameter of not lessthan 5 nm and not greater than 70 nm. In some embodiments, theorganic-inorganic hybrid polymer dots disclosed herein can have adiameter of not less than 5 nm and not greater than 60 nm. In someembodiments, the organic-inorganic hybrid polymer dots disclosed hereincan have a diameter of not less than 5 nm and not greater than 40 nm. Insome embodiments, the organic-inorganic hybrid polymer dots disclosedherein can have a diameter of not less than 5 nm and not greater than 30nm. In some embodiments, the organic-inorganic hybrid polymer dotsdisclosed herein can have a diameter of not less than 5 nm and notgreater than 25 nm.

The attributes of the organic-inorganic hybrid polymer dots can beadjusted as desired in order to tune a variety of photophysicalproperties (e.g., absorbance, emission brightness, and/or the wavelengthof maximum emission). Notably, in some cases, quenching of fluorescenceis not increased due to particle formation. It will be appreciated thatpolymer dots having high brightness and specific binding capabilitiesprovide important attributes to advance imaging and detection techniquesfor studying chemical and biological analytes and systems. In someembodiments, the organic-inorganic hybrid polymer dots disclosed hereincan have a quantum yield of at least 1%. In some embodiments, theorganic-inorganic hybrid polymer dots disclosed herein can have aquantum yield at least 5%. In some embodiments, the quantum yield ismore than 5%, more than 10%, more than 20%, more than 30%, more than40%, more than 50%, more than 60%, more than 70%, more than 80%, or morethan 90%.

Organic-Inorganic Hybrid Polymer Dots with a Siloxane Network

In some embodiments, the hybrid polymer dots described herein include asiloxane network, e.g., a network including a plurality of Si—O—Silinkages. A siloxane network can be formed by the full or partialhydrolysis of one or more silane and/or siloxane species. For example,in certain embodiments, a siloxane network is fully or partiallyhydrolyzed from an alkyl silane, an alkoxy silane, a chloro silane, anorthosilicate, a siloxane, an alpha silane, an acetoxy silane, an aminosilane, a bis silane, an epoxy silane, a halo silane, a hydrogen silane,a hydrogen siloxane, a hydroxyl silane, an ester silane, an aryl silane,an acryl silane, a methacryl silane, a styryl silane, a vinyl silane, anolefin silane, a sulfur silane, a phosphine silane, a phosphate silane,an isocyanate silane, an azide silane, an anhydride silane, or acombination thereof. In certain embodiments, the siloxane network isfully or partially hydrolyzed from octodecyltrimethoxysilane,octodecyltrichlorosilane, tetraethylorthosilicate,trifluoropropyltrimethoxysilane, phenyltrimethoxysilane,chloropropyltrimethoxysilane, heptadecafluorodecyltrichlorosilane,glycidoxypropyltrimethoxysilane, epoxyhexyltriethoxysilane,hydroxymethyltriethoxysilane, iodopropyltrimethoxysilane,isocyantopropyltrimethoxysilane, methacryloxymethyltriethoxysilane,vinyltrimethoxysilane, styrylethyltrimethoxysilane, or a combinationthereof. In certain embodiments, the siloxane network is fully orpartially hydrolyzed from octodecyltrimethoxysilane,octodecyltrichlorosilane, or tetraethylorthosilicate, or a combinationthereof.

The weight percent of the siloxane network and/or the components thereof(e.g., silicon) in a hybrid polymer dot can be varied as desired. Insome embodiments, the weight percent of the siloxane network and/or thecomponents thereof (e.g., silicon) is selected to avoid formation of acore-shell structure in the resulting hybrid polymer dot. In certainembodiments, the weight percent of silicon from the siloxane network inthe hybrid polymer dot is less than or equal to about 1%, less than orequal to about 5%, less than or equal to about 10%, less than or equalto about 15%, less than or equal to about 20%, less than or equal toabout 25%, less than or equal to about 30%, less than or equal to about35%, less than or equal to about 40%, less than or equal to about 45%,or less than or equal to about 47%. In certain embodiments, the weightpercent of silicon from the siloxane network in the hybrid polymer dotis greater than or equal to about 1%, greater than or equal to about 5%,greater than or equal to about 10%, greater than or equal to about 15%,greater than or equal to about 20%, greater than or equal to about 25%,greater than or equal to about 30%, greater than or equal to about 35%,greater than or equal to about 40%, or greater than or equal to about45%. In certain embodiments, the weight percent of silicon from thesiloxane network in the hybrid polymer dot is within a range from about1% to about 45%, or within a range from about 1% to about 47%.

Organic-inorganic hybrid polymer dots can comprise at least two silanespecies, each having their own respective function. For example, theorganic-inorganic hybrid polymer dots can have a surface that isfunctionalized with a functional silane species comprising a carboxyl,an amine, a thiol (—SH), a carboxylate or carboxylic acid, a maleimide,a maleic anhydride, a N-hydroxysuccinimide (NHS), an alcohol (—OH), or acyanate, or a combination thereof that is suitable for bioconjugation.Additionally, hybrid polymer dots can comprise an aliphatic chain, forexample, an alkyl chain. The aliphatic chain can take part in crosslinking during formation of the interpenetrated network. The aliphaticchain can be physically associated with the organic network (e.g.,chromophoric polymer) and/or the siloxane network during formation ofthe interpenetrated network.

The hybrid polymer dot can comprise a siloxane network and at least oneother network to form the interpenetrated organic-inorganic network. Forexample, in some embodiments, the present disclosure providesorganic-inorganic hybrid polymer dots comprising a semiconductingchromophoric polymer and a siloxane network, wherein the semiconductingchromophoric polymer and the siloxane network form an organic-inorganicinterpenetrated network. The interpenetrated network can be a mesh-likeand/or interlocking structure of the siloxane network interpenetratedwith the chromophoric polymer (e.g., without forming a core-cap or acore-shell structure).

An organic-inorganic hybrid polymer dot with a siloxane network can beformed in various ways. In certain embodiments, the hybrid polymer dotis formed through the physical association of the siloxane network withthe chromophoric polymer so as to form an interpenetrated network. Incertain embodiments, the hybrid polymer dot is formed wherein thesiloxane network comprises an alkyl chain and wherein the semiconductingchromophoric polymer is physically associated with an alkyl chain of thesiloxane network, thereby forming the organic-inorganic interpenetratednetwork. Alternatively or in combination, the siloxane network andchromophoric polymer can be chemically bonded (e.g., covalently bonded)to each other to form the interpenetrated network.

Hybrid Polymer Dots with Physical Association Between a Siloxane Networkand a Semiconducting Chromophoric Polymer

In some embodiments, the present disclosure provides hybrid polymer dotsin which the siloxane network is physically associated with thesemiconducting chromophoric polymer, such as by hydrophobic interaction.For example, the siloxane network can comprise an aliphatic chain andthe semiconducting chromophoric polymer can be physically associatedwith the aliphatic chain of the siloxane network, thereby forming theorganic-inorganic interpenetrated network. The aliphatic chain cancomprise at least 5, at least 10, at least 15, or at least 20 carbons.The aliphatic chain can comprise, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17, at least 18, atleast 19, or at least 20 carbons. In some embodiments, the aliphaticside chain comprises at least 10 carbons. In some embodiments, thealiphatic chain is an alkyl chain. The alkyl chain can comprise at least5, at least 10, at least 15, or at least 20 carbons. The alkyl chain cancomprise, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18, at least 19, or at least 20carbons. In some embodiments, the alkyl chain comprises at least 10carbons. In some embodiments, the siloxane network comprises analkylene, alkoxy, alkenyl, alkenylene, alkynyl, alkynylene, alkyl amine,cycloalkyl, cycloalkylene, heterocycloalkyl, or heterocycloalkylene.

In some embodiments, the siloxane network includes one or moreorthogonally cross-linked units. In certain embodiments, an orthogonallycross-linked unit includes a reactive group that cross-links only withits designated reactive group, but not with another reactive group thatmay also be present. For example, reactive groups A and B can form adesignated pair that cross-link with each other, and reactive groups Yand Z can form another designated pair that cross-link with each other.In such embodiments, reactive group A is considered to be orthogonalwith respect to Y because A does not cross-link with Z, and reactivegroup Y is orthogonal with respect to A because Y does not cross-linkwith B. In some embodiments, reactive groups A can cross-link with eachother or with reactive groups B to form a siloxane network, and reactivegroups Y do not cross-link with either A or B, such that A and Y, and/orB and Y, are considered to be orthogonal cross-linking units.

In some embodiments, the siloxane network can comprise a plurality ofinterconnected units, and each interconnected unit can be selected fromthe group consisting of:

wherein: A⁰, A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹ are each independentlyC_(n)F_(2n+1), C_(n)H_(2n)X, C_(n)F_(2n+1), or C_(n)F_(2n)X; wherein Xis a functional group suitable for bioconjugation; wherein D⁰, D¹, D²,D³, D⁴, D⁵, D⁶, D⁷, D⁸, D⁹ are each independently LX, wherein L is alinker moiety; and n is not less than 1. In some embodiments, n is notless than 2, not less than 3, not less than 4, not less than 5, not lessthan 6, not less than 7, not less than 8, not less than 9, or not lessthan 10. In some embodiments, n is not less than 6. In some embodiments,n is not greater than 20. In some embodiments, n is not greater than 40.In some embodiments, n is not greater than 60. In some embodiments, n isnot less than 6 and is not greater than 20. In some embodiments, n isnot less than 6 and is not greater than 15. In some embodiments, n isnot less than 6 and is not greater than 10. Functional groups suitablefor bioconjugation, also represented by “X,” linker moieties, alsorepresented by “L,” and the combination thereof, also represented by “D”and “LX,” are described in further detail herein below.

In some embodiments, the chromophoric polymer is physically associatedwith but not covalently bonded to the siloxane network. For example, invarious embodiments, the chromophoric polymer is not silanefunctionalized, and functionalization and formation of theinterpenetrated network of the hybrid polymer dot is achieved by thephysical association (e.g., hydrophobic interaction) of the chromophoricpolymer with the siloxane network only. In alternative embodiments, thechromophoric polymer can also be covalently bonded with the siloxanenetwork, as discussed in greater detail below herein.

Hybrid Polymer Dots with Covalent Bonding Between a Siloxane Network aSemiconducting Chromophoric Polymer

In some embodiments, the present disclosure provides hybrid polymer dotsin which the siloxane network is covalently bonded with thesemiconducting chromophoric polymer. For example, a hybrid polymer dotcan comprise a semiconducting chromophoric polymer and a siloxanenetwork that is covalently bonded to the semiconducting chromophoricpolymer. Optionally, the hybrid polymer dot can also include afunctional group (X) suitable for bioconjugation. In certainembodiments, the siloxane network is also physically associated with thechromophoric polymer in order to form an organic-inorganicinterpenetrated network.

In some embodiments, the chromophoric polymer is silane functionalized.Functionalization of the hybrid polymer dot and formation of theinterpenetrated network can be achieved by the presence of at least twosilane species on the chromophoric polymer, each having its ownrespective function. The at least two silane species can be present aspendant side chains on the chromophoric polymer. The pendant side chainson the chromophoric polymer can include a silane chain (“R^(S)”) and afunctional silane chain including one or more functional groups(“R^(F)”). as shown below. One of the at least two silane species (e.g.,the silane chain or functional silane chain) can comprise an aliphaticchain. The aliphatic chain can comprise at least 5, at least 10, atleast 15, or at least 20 carbons. The aliphatic chain can comprise, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 carbons. Insome embodiments, the aliphatic side chain comprises at least 10carbons. In some embodiments, the aliphatic chain is an alkyl chain. Thealkyl chain can comprise at least 5, at least 10, at least 15, or atleast 20 carbons. The alkyl chain can comprise, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, or at least 20 carbons. In some embodiments, thealkyl chain comprises at least 10 carbons. In some embodiments, thesiloxane network comprises an alkylene, alkoxy, alkenyl, alkenylene,alkynyl, alkynylene, alkyl amine, cycloalkyl, cycloalkylene,heterocycloalkyl, heterocycloalkylene. The alkyl chain can take part incross linking during formation of the interpenetrated network. The alkylchain can be chemically associated with the chromophoric polymer and/orthe siloxane network during formation of the interpenetrated network.Additionally, the chromophoric polymer can comprise a functional silanespecies comprising a carboxyl, an amine, a thiol (—SH), a carboxylate orcarboxylic acid, a maleimide, a maleic anhydride, a N-hydroxysuccinimide(NHS), an alcohol (—OH), or a cyanate, or a combination thereof that issuitable for bioconjugation. In some embodiments, the chromophoricpolymer can comprise a carboxyl. In some embodiments, the hybrid polymerdots have a surface that is functionalized with the functional silanespecies that is suitable for bioconjugation.

FIG. 12 provides a general schematic illustration of conjugated polymerswith a silane chain and functional silane chain for bioconjugation. Insome embodiments, a semiconducting chromophoric polymer can comprise aplurality of units, M, which can be selected from:

wherein:

-   -   R^(S) is

-   -   R^(F) is

A⁰, A⁴, A⁸, A¹², A¹⁵, A¹⁸, are each independently C_(n)H_(2n) orC_(n)F_(2n); A¹, A², A³, A⁵, A⁶, A⁷, A⁹, A¹⁰, A¹¹, A¹³, A¹⁴, A¹⁶, A¹⁷,A¹⁹ are each independently C_(m)H_(2m+1) or C_(m)H_(2m+1), C_(m)F_(2m),X¹, X², X³, X⁴ are each independently a functional group containing oneor more active groups including but not limited to an amine, acarboxylate or carboxylic acid, a maleimide, a maleic anhydride, a thiol(—SH), a N-hydroxysuccinimide (NHS), or any of the other functionalgroups described herein; n is not less than 1; and m is not less than 1.In some embodiments, n is not less than 2, is not less than 3, is notless than 4, is not less than 5, is not less than 6, is not less than 7,is not less than 8, is not less than 9, or is not less than 10. In someembodiments, n is not less than 2. In some embodiments, n is not greaterthan 40. In some embodiments, n is not greater than 20. In someembodiments, n is not less than 1 and is not greater than 20. In someembodiments, n is not less than 2 and is not greater than 20. In someembodiments, m is not less than 2, not less than 3, not less than 4, notless than 5, not less than 6, not less than 7, not less than 8, not lessthan 9, or not less than 10. In some embodiments, m is not less than 3.In some embodiments, m is not less than 6. In some embodiments, m is notgreater than 5, not greater than 6, not greater 7, not greater than 8,not greater than 9, or not greater than 10. In some embodiments, m isnot greater than 20. In some embodiments, m is not greater than 40. Insome embodiments, m is not greater than 60.

In some embodiments, the pendant side chains on the chromophoric polymerinclude a silane chain (“R^(S)”) and a functional chain including one ormore functional groups (“LX”), as shown below. The silane chain cancomprise an aliphatic chain. The aliphatic chain can comprise at least5, at least 10, at least 15, or at least 20 carbons. The aliphatic chaincan comprise, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, or atleast 20 carbons. In some embodiments, the aliphatic side chaincomprises at least 10 carbons. In some embodiments, the aliphatic chainis an alkyl chain. The alkyl chain can comprise at least 5, at least 10,at least 15, or at least 20 carbons. The alkyl chain can comprise, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 carbons. Insome embodiments, the alkyl chain comprises at least 10 carbons. In someembodiments, the siloxane network comprises an alkylene, alkoxy,alkenyl, alkenylene, alkynyl, alkynylene, alkyl amine, cycloalkyl,cycloalkylene, heterocycloalkyl, heterocycloalkylene. The alkyl chaincan take part in cross linking during formation of the interpenetratednetwork. The alkyl chain can be chemically associated with thechromophoric polymer and/or the siloxane network during formation of theinterpenetrated network. Additionally, the chromophoric polymer cancomprise a functional chain including a functional group (“X”)comprising a carboxyl, an amine, a thiol (—SH), a carboxylate orcarboxylic acid, a maleimide, a maleic anhydride, a N-hydroxysuccinimide(NHS), an alcohol (—OH), or a cyanate, or a combination thereof that issuitable for bioconjugation. The functional group can be connected tothe polymer backbone via a linker (“L”). In some embodiments, thechromophoric polymer can comprise a carboxyl. In some embodiments, thehybrid polymer dots have a surface that is functionalized with thefunctional species that is suitable for bioconjugation.

FIG. 13 provides a general schematic illustration of conjugated polymerswith a silane chain and functional chain for bioconjugation. In someembodiments, a semiconducting chromophoric polymer can comprise aplurality of units, M, which can be selected from:

wherein:

-   -   R^(S) is

A⁰, A⁴, A⁸, are each independently C_(n)H_(2n) or C_(n)F_(2n); A¹, A²,A³, A⁵, A⁶, A⁷, A⁹, A¹⁰, A¹¹, are each independently C_(m)H_(2m+1) orC_(m)F_(2m+1), C_(m)F_(2m); X is a functional group containing one ormore active groups including but not limited to an amine, a carboxylateor carboxylic acid, a maleimide, a maleic anhydride, a thiol (—SH), aN-hydroxysuccinimide (NHS), or any of the other functional groupsdescribed herein; L is a linker between the polymer backbone and thefunctional group X; n is not less than 1; and m is not less than 1. Insome embodiments, n is not less than 2, is not less than 3, is not lessthan 4, is not less than 5, is not less than 6, is not less than 7, isnot less than 8, is not less than 9, or is not less than 10. In someembodiments, n is not less than 2. In some embodiments, n is not greaterthan 40. In some embodiments, n is not greater than 20. In someembodiments, n is not less than 1 and is not greater than 20. In someembodiments, m is not less than 2, not less than 3, not less than 4, notless than 5, not less than 6, not less than 7, not less than 8, not lessthan 9, or not less than 10. In some embodiments, n is not less than 2and is not greater than 20. In some embodiments, m is not less than 3.In some embodiments, m is not less than 6. In some embodiments, m is notgreater than 5, not greater than 6, not greater 7, not greater than 8,not greater than 9, or not greater than 10. In some embodiments, m isnot greater than 20. In some embodiments, m is not greater than 40. Insome embodiments, m is not greater than 60. In some embodiments, m isnot less than 1 and is not greater than 20.

In some embodiments, the pendant side chains on the chromophoric polymerinclude a functional silane chain (“R^(F)”) including one or morefunctional groups, as shown below. The functional silane chain cancomprise an aliphatic chain. The aliphatic chain can comprise at least5, at least 10, at least 15, or at least 20 carbons. The aliphatic chaincan comprise, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, or atleast 20 carbons. In some embodiments, the aliphatic side chaincomprises at least 10 carbons. In some embodiments, the aliphatic chainis an alkyl chain. The alkyl chain can comprise at least 5, at least 10,at least 15, or at least 20 carbons. The alkyl chain can comprise, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, or at least 20 carbons. Insome embodiments, the alkyl chain comprises at least 10 carbons. In someembodiments, the siloxane network comprises an alkylene, alkoxy,alkenyl, alkenylene, alkynyl, alkynylene, alkyl amine, cycloalkyl,cycloalkylene, heterocycloalkyl, heterocycloalkylene. The alkyl chaincan take part in cross linking during formation of the interpenetratednetwork. The alkyl chain can be chemically associated with thechromophoric polymer and/or the siloxane network during formation of theinterpenetrated network. Additionally, the functional silane chain caninclude a functional group (“X”) comprising a carboxyl, an amine, athiol (—SH), a carboxylate or carboxylic acid, a maleimide, a maleicanhydride, a N-hydroxysuccinimide (NHS), an alcohol (—OH), or a cyanate,or a combination thereof that is suitable for bioconjugation. In someembodiments, the hybrid polymer dots have a surface that isfunctionalized with the functional species that is suitable forbioconjugation.

FIG. 14 provides a general schematic illustration of conjugated polymerswith a silane chain and functional chain for bioconjugation. In someembodiments, a semiconducting chromophoric polymer can comprise aplurality of units, M, which can be selected from:

wherein:

-   -   R^(F) is

A⁰, A³, A⁶ are each independently C_(n)H_(2n) or C_(n)F_(2n); A¹, A²,A⁴, A⁵, A⁷ are each independently C_(m)H_(2m+1) or C_(m)F_(2m+1); X¹,X², X³, X⁴ are each independently a functional group containing one ormore active groups including but not limited to an amine, a carboxylateor carboxylic acid, a maleimide, a maleic anhydride, a thiol (—SH), aN-hydroxysuccinimide (NHS), or any of the other functional groupsdescribed herein; n is not less than 1; and m is not less than 1. Insome embodiments, n is not less than 2, is not less than 3, is not lessthan 4, is not less than 5, is not less than 6, is not less than 7, isnot less than 8, is not less than 9, or is not less than 10. In someembodiments, n is not less than 2. In some embodiments, n is not greaterthan 40. In some embodiments, n is not greater than 20. In someembodiments, n is not less than 1 and is not greater than 20. In someembodiments, n is not less than 2 and is not greater than 20. In someembodiments, m is not less than 2, not less than 3, not less than 4, notless than 5, not less than 6, not less than 7, not less than 8, not lessthan 9, or not less than 10. In some embodiments, m is not less than 3.In some embodiments, m is not less than 6. In some embodiments, m is notgreater than 5, not greater than 6, not greater 7, not greater than 8,not greater than 9, or not greater than 10. In some embodiments, m isnot greater than 20. In some embodiments, m is not greater than 40. Insome embodiments, m is not greater than 60. In some embodiments, m isnot less than 1 and is not greater than 20.

In some embodiments, the semiconducting chromophoric polymer cancomprise a functional group suitable for bioconjugation (X) and analkoxylsilyl or alkylsilyl.

In some embodiments, the siloxane network can comprise a plurality ofinterconnected units, wherein the plurality of interconnected units cancomprise a unit selected from the group consisting of:

wherein: A⁰, A¹, A², A³ are each independently C_(p)H_(2p+1),C_(p)F_(2p+1), C_(p)H_(2p)X, or C_(p)F_(2p)X; D is LX, wherein L is alinker moiety; and p is not less than 1. In some embodiments, p is notless than 2, p is not less than 3, p is not less than 4, not less than5, not less than 6, not less than 7, not less than 8, not less than 9,or not less than 10. In some embodiments, p is not less than 6. In someembodiments, p is not greater than 20. In some embodiments, p is notgreater than 40. In some embodiments, p is not greater than 60. In someembodiments, p is not less than 6 and is not greater than 20. In someembodiments, p is not less than 6 and is not greater than 15. In someembodiments, p is not less than 6 and is not greater than 10.

In some embodiments, an organo-silane is used to form a hybrid polymerdot including a siloxane network, and the weight percent of the siloxanenetwork and/or the components thereof (e.g., silicon) in the hybridpolymer dot can be varied as desired. In some embodiments, the weightpercent of the siloxane network and/or the components thereof (e.g.,silicon) is selected to avoid formation of a core-shell structure in theresulting hybrid polymer dot. In certain embodiments, the weight percentof silicon from the siloxane network in the hybrid polymer dot is lessthan or equal to about 1%, less than or equal to about 5%, less than orequal to about 10%, less than or equal to about 15%, less than or equalto about 20%, less than or equal to about 25%, less than or equal toabout 30%, less than or equal to about 35%, less than or equal to about40%, less than or equal to about 45%, or less than or equal to about47%. In certain embodiments, the weight percent of silicon from thesiloxane network in the hybrid polymer dot is greater than or equal toabout 1%, greater than or equal to about 5%, greater than or equal toabout 10%, greater than or equal to about 15%, greater than or equal toabout 20%, greater than or equal to about 25%, greater than or equal toabout 30%, greater than or equal to about 35%, greater than or equal toabout 40%, or greater than or equal to about 45%. In certainembodiments, the weight percent of silicon from the siloxane network inthe hybrid polymer dot is within a range from about 1% to about 45%, orwithin a range from about 1% to about 47%.

In some embodiments, the polymer dot can comprise at least twoorthogonal reactive chemical groups. In some embodiments, one of the atleast two orthogonal reactive chemical groups that the polymer dotcomprises has the formula C_(n)H_(2n)X or C_(n)F_(2n)X, wherein X is afunctional group suitable for bioconjugation and n is not less than 1.In some embodiments, one of the at least two orthogonal reactivechemical groups of the polymer dot comprises X, where X is a functionalgroup suitable for bioconjugation. The semiconducting chromophoricpolymer can comprise at least two orthogonal reactive chemical groups.In some embodiments, one of the at least two orthogonal reactivechemical groups that the semiconducting chromophoric polymer compriseshas the formula C_(n)H_(2n)X or C_(n)F_(2n)X, wherein X is a functionalgroup suitable for bioconjugation and n is not less than 1. In someembodiments, n is not less than 2, n is not less than 3, n is not lessthan 4, not less than 5, not less than 6, not less than 7, not less than8, not less than 9, or not less than 10. In some embodiments, n is notless than 6. In some embodiments, n is not greater than 20. In someembodiments, n is not greater than 40. In some embodiments, n is notgreater than 60. In some embodiments, n is not less than 6 and is notgreater than 20. In some embodiments, n is not less than 6 and is notgreater than 15. In some embodiments, n is not less than 6 and is notgreater than 10.

Chromophoric Polymers for Use in Hybrid Polymer Dots

The hybrid polymer dots described herein can comprise various types ofchromophoric polymers, such as one or more of the chromophoric polymertypes described herein. Hybrid polymer dots can include one or morechromophoric polymers (e.g., semiconducting chromophoric polymers) thathave been collapsed into a stable sub-micron sized particle.

In some embodiments, the hybrid polymer dots of the present disclosurecomprise a plurality of polymers. In certain embodiments the polymer dotcan comprise a plurality of semiconducting chromophoric polymers. Incertain embodiments, the hybrid polymer dots can comprise a blend ofchromophoric polymers and non-chromophoric polymers. In certainembodiments, the hybrid polymer dots can comprise a blend ofsemiconducting chromophoric polymers. In certain embodiments, the hybridpolymer dots include a blend of semiconducting chromophoric polymers.The blends can include any combination of homopolymers, copolymers, andoligomers. Polymer blends used to form hybrid polymer dots may beselected in order to tune the properties of the resulting polymerparticles, for example, to achieve a desired excitation or emissionspectra for the hybrid polymer dot.

The hybrid polymer dots can comprise polymers with one or more repeatingunits, which can be combined in fixed, ordered, or random configurationsand ratios. A repeating unit can be a monomer or a chemical motif thatoccurs throughout the polymer, such as an aromatic or heterocyclic unit.The polymers can be halogenated, for example, fluorinated, chlorinated,brominated, or iodinated. A polymer, a repeating unit, or a monomer canbe halogenated at one or multiple sites. A halogenated polymer, forexample, a fluorinated polymer, can provide greater levels offluorescence than can a non-halogenated analogous polymer.

Any suitable number and combination of chromophoric polymer types can beincorporated in the hybrid polymer dots described herein, such as one ormore chromophoric polymers, two or more chromophoric polymers, three ormore chromophoric polymers, four or more chromophoric polymers, five ormore chromophoric polymers, six or more chromophoric polymers, seven ormore chromophoric polymers, eight or more chromophoric polymers, nine ormore chromophoric polymers, ten or more chromophoric polymers, fifty ormore chromophoric polymers, or one hundred or more chromophoricpolymers.

The chromophoric polymer can be a homopolymer or a heteropolymer. Invarious embodiments, the chromophoric polymer can be a semiconductingpolymer, a non-semiconducting polymer, or a combination thereof. Forexample, a number of semiconducting polymers are suitable for use inhybrid polymer dots according to the present disclosure. Semiconductingpolymers have been developed with emission wavelengths ranging from UVto infrared, including the entire visible spectrum. Examples ofsemiconducting polymers include but are not limited to:polyfluorene-based polymers, including but not limited topoly(9,9-dihexylfluorenyl-2,7-diyl) (PDHF)-based andpoly(9,9-dioctylfluorenyl-2,7-diyl) (PFO)-based polymers; fluorene-basedcopolymers, including but not limited to,poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}](PFPV)-based,poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1,3}-thiadiazole)](PFBT)-based,poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-Di-2-thienyl-2,1,3-benzothiadiazole)](PFTBT)-based, andpoly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-Di-2-thienyl-2,1,3-benzothiadiazole)](PF-0.1TBT)-basedpolymers; phenylene vinylene-based polymers, including but not limitedto, poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene](MEH-PPV)-based andpoly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(l-cyanovinylene-1,4-phenylene)](CN-PPV)-based polymers; phenylene ethynylene-based polymers, includingbut not limited to,poly(2,5-di(3′,7′-dimethyloctyl)phenylene-1,4-ethynylene (PPE)-basedpolymers; or a combination thereof.

A wide variety of chromophoric polymer structures are suitable for usein accordance with various embodiments and embodiments of the presentdisclosure. In some embodiments, the chromophoric polymer is a linearpolymer. In other embodiments, the chromophoric polymer is a branchedpolymer. In certain embodiments, the chromophoric polymer is adendrimer. In certain embodiments, the chromophoric polymer is a brushpolymer. In certain embodiments, the chromophoric polymer is a starpolymer.

In some embodiments, the chromophoric polymers contain a polymerfunctionalized on the terminal monomeric unit, for example with acarboxyl, amine, thiol, ester, succinimidyl ester, azide, alkyne,cyclooctyne, phosphine, or similar functional group. Examples of suchpolymers include but are not limited to poly(meth)acrylate polymers,polyacrylamide polymers, polyisobutylene, polydiene, polyphenylene,polyethylene, polyethylene glycol), polylactide, polystyrene,polysiloxane, poly(vinyl pyridine), poly(vinylpyrrolidone),polyurethane, a block copolymer thereof, a random or alternatingcopolymer thereof, and the like.

In some embodiments of the present disclosure, the hybrid polymer dotsprovided herein include the polymer CN-PPV, also known aspoly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(l-cyanovinylene-1,4-phenylene)],which is a bright, compact, and orange-light-emitting semiconductingpolymer particle. In certain embodiments, CN-PPV has superiorfluorescence properties, such as a large absorption cross-section, highquantum yield, and a fast emission rate. In some embodiments, the hybridpolymer dot comprises a polymer that consists essentially of CN-PPV. Insome embodiments, the particle includes CN-PPV and at least one othermaterial. For example, the CN-PPV can form part of a copolymer or bemixed with a copolymer or other material that provides an additionalfunctionality.

In some embodiments, the hybrid polymer dots of the present disclosureinclude a semiconducting copolymer having at least two differentchromophoric units. For example, a conjugated or semiconductingcopolymer can contain both fluorene and benzothiazole chromophoric unitspresent at a given ratio. Typical chromophoric units used to synthesizesemiconducting copolymers include, but are not limited to fluoreneunits, phenylene vinylene units, phenylene units, phenylene ethynyleneunits, benzothiazole units, thiophene units, carbazole fluorene units,boron-dipyrromethene units, squaraine units, and derivatives thereof.The different chromophoric units can be segregated, as in a blockcopolymer, or intermingled. In some embodiments, a chromophoriccopolymer is represented by writing the identity of the majorchromophoric species. For example, PFBT is a chromophoric polymercontaining fluorene and benzothiazole units at a certain ratio. In someembodiments, a dash is used to indicate the percentage of the minorchromophoric species and then the identity of the minor chromophoricspecies. For example, PF-0.1 BT is a chromophoric copolymer containing90% PF and 10% BT.

In some embodiments, the hybrid polymer dots of the present disclosureinclude polymers that have a narrow band emissive unit (e.g., a narrowband monomer and/or a narrow band unit). For example, the presentdisclosure can include a homopolymer or heteropolymer including a narrowband monomer, such as BODIPY and/or BODIPY derivative monomer, asquaraine and/or squaraine derivative monomer, a metal complex and/ormetal complex derivative monomer, a porphyrin and/or porphyrinderivative monomer, a metalloporphyrin and/or metalloporphyrinderivative monomer, a phthalocyanine and/or phthalocynanine derivativemonomer, a lanthanide complex and/or lanthanide complex derivativemonomer, a perylene and/or perylene derivative monomer, a cyanine and/orcyanine derivative monomer, a rhodamine and/or rhodamine derivativemonomer, a coumarin and/or coumarin derivative monomer, and/or axanthene and/or xanthene derivative monomer. In certain embodiments, anarrow band unit is, e.g., a narrow band monomer or a fluorescentnanoparticle embedded in or attached to the polymer dot.

In some embodiments, the hybrid polymer dots can comprise a fluorenepolymer, a fluorene-based polymer or copolymer, a phenylenevinylene-based polymer or copolymer, a phenylene ethynylene-basedpolymer or copolymer, and a BODIPY-based polymer or copolymer. In someembodiments, the hybrid polymer dots can comprisepoly(9,9-dihexylfluorenyl-2,7-diyl) (PDHF),Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO),poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}](PFPV),poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1,3}-thiadiazole)](PFBT),poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-Di-2-thienyl-2,1,3-benzothiadiazole)](PFTBT),poly[(9,9-dioctylfluorenyl-2,7-diyl)-9-co-(4,7-Di-2-thienyl-2,1,3-benzothiadiazole)](PF-0.1TBT)), poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene](MEH-PPV),poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(l-cyanovinylene-1,4-phenylene)](CN-PPV), BODIPY 570, BODIPY 590, or BODIPY 690.

In some embodiments, the hybrid polymer dots can comprise a BODIPYderivative. The BODIPY derivative can have the structure of Formula (I):

wherein each of R¹, R^(2A), R^(2B), R^(3A), R^(3B), R^(4A) and R^(4B) isindependently selected from the group consisting of, but not limited to,hydrogen (H), deuterium (D), halogen, direct or branched alkyl,heteroalkyl, heterocycloalkyl, heterocycloalkylene, alkoxy, aryl,hydroxyl, cyano, nitro, ether and its derivatives, ester and itsderivatives, alkyl ketone, alkylester, arylester, alkynyl, alkyl amine,fluoroalkyl, fluoroaryl, and polyalkalene (e.g., methoxy ethoxy ethoxy,ethoxy ethoxy, and —(OCH₂CH₂)_(n)OH, n=1-50), phenyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted phenyl, pyridyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyridyl,bipyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbipyridyl tripyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted tripyridyl, furyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted furyl, thienyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted thienyl, pyrrolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrrolyl,pyrazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazolyl, oxazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted oxazolyl, thiazolyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted thiazolyl, imidazolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted imidazolyl,pyrazinyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazinyl, benzooxadizolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted benzooxadizolyl, benzothiadizolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbenzothiadizolyl, fluorenyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted fluorenyl, triphenylaminyl-substitutedfluorenyl, diphenylaminyl-substituted fluorenyl, alkyl-substitutedcarbazolyl, alkyl-substituted triphenylaminyl and alkyl-substitutedthiophenyl. As exemplary embodiments, alkyl substituted phenyl caninclude 2-alkylphenyl, 3-alkylphenyl, 4-alkylphenyl, 2,4-dialkylphenyl,3,5-dialkylphenyl, 3,4-dialkylphenyl; alkyl-substituted fluorenyl caninclude 9, 9-dialkyl-substituted fluorenyl,7-alkyl-9,9-dialkyl-substituted fluorenyl,6-alkyl-9,9-dialkyl-substituted fluorenyl,7-triphenylaminyl-9,9-dialkyl-substituted fluorenyl and7-diphenylaminyl-9,9-dialkyl-substituted fluorenyl; alkyl-substitutedcarbazolyl can include N-alkyl-substituted carbazolyl,6-alkyl-substituted carbazolyl and 7-alkyl-substituted carbazolyl;alkyl-substituted triphenylaminyl can include 4′-alkyl-substitutedtriphenylaminyl, 3′-alkyl-substituted triphenylaminyl,3′,4′-dialkyl-substituted triphenylaminyl and 4′,4″-alkyl-substitutedtriphenylaminyl; alkyl-substituted thiophenyl can include2-alkylthiophenyl, 3-alkylthiophenyl, and 4-alkylthiophenyl,N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, and N-dialkoxyphenyl-4-phenyl.The narrow-band monomer can be integrated into a backbone of the polymer(e.g., polymerized in the polymer) and/or covalently attached to thebackbone, a terminus, or a sidechain of the polymer through at least oneattachment to R¹, R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), or acombination thereof.

In some embodiments, the hybrid polymer dots of the present disclosureinclude a polymer that includes a narrow-band monomer having thestructure of Formula (II):

wherein each of R¹, R^(2A), R^(2B), R^(3A), R^(3B), R^(4A) and R^(4B) isindependently selected from the group consisting of, but not limited to,hydrogen (H), deuterium (D), halogen, direct or branched alkyl,heteroalkyl, heterocycloalkyl, heterocycloalkylene, alkoxy, aryl,hydroxyl, cyano, nitro, ether and its derivatives, ester and itsderivatives, alkyl ketone, alkylester, arylester, alkynyl, alkyl amine,fluoroalkyl, fluoroaryl, and polyalkalene (e.g., methoxy ethoxy ethoxy,ethoxy ethoxy, and —(OCH₂CH₂)_(n)OH, n=1-50), phenyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted phenyl, pyridyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyridyl,bipyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbipyridyl tripyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted tripyridyl, furyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted furyl, thienyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted thienyl, pyrrolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrrolyl,pyrazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazolyl, oxazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted oxazolyl, thiazolyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted thiazolyl, imidazolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted imidazolyl,pyrazinyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazinyl, benzooxadizolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted benzooxadizolyl, benzothiadizolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbenzothiadizolyl, fluorenyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted fluorenyl, triphenylaminyl-substitutedfluorenyl, diphenylaminyl-substituted fluorenyl, alkyl-substitutedcarbazolyl, alkyl-substituted triphenylaminyl and alkyl-substitutedthiophenyl. As exemplary embodiments, alkyl substituted phenyl caninclude 2-alkylphenyl, 3-alkylphenyl, 4-alkylphenyl, 2,4-dialkylphenyl,3,5-dialkylphenyl, 3,4-dialkylphenyl; alkyl-substituted fluorenyl caninclude 9, 9-dialkyl-substituted fluorenyl,7-alkyl-9,9-dialkyl-substituted fluorenyl,6-alkyl-9,9-dialkyl-substituted fluorenyl,7-triphenylaminyl-9,9-dialkyl-substituted fluorenyl and7-diphenylaminyl-9,9-dialkyl-substituted fluorenyl; alkyl-substitutedcarbazolyl can include N-alkyl-substituted carbazolyl,6-alkyl-substituted carbazolyl and 7-alkyl-substituted carbazolyl;alkyl-substituted triphenylaminyl can include 4′-alkyl-substitutedtriphenylaminyl, 3′-alkyl-substituted triphenylaminyl,3′,4′-dialkyl-substituted triphenylaminyl and 4′,4″-alkyl-substitutedtriphenylaminyl; alkyl-substituted thiophenyl can include2-alkylthiophenyl, 3-alkylthiophenyl, and 4-alkylthiophenyl,N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, and N-dialkoxyphenyl-4-phenyl.The narrow-band monomer can be integrated into a backbone of the polymer(e.g., polymerized in the polymer) and/or covalently attached to thebackbone, a terminus, or a sidechain of the polymer through at least oneattachment to R¹, R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), or acombination thereof. The monomer can, for example, integrate with thebackbone of the polymer by attachment to the R^(3A) and R^(3B) groups.

In some embodiments, the hybrid polymer dots of the present disclosureinclude a polymer that includes a narrow-band monomer having thestructure of Formula (III):

wherein each of R¹, R^(2A) and R^(2B) is independently selected from thegroup consisting of, but not limited to, hydrogen (H), deuterium (D),halogen, direct or branched alkyl, heteroalkyl, heterocycloalkyl,heterocycloalkylene, alkoxy, aryl, hydroxyl, cyano, nitro, ether and itsderivatives, ester and its derivatives, alkyl ketone, alkylester,arylester, alkynyl, alkyl amine, fluoroalkyl, fluoroaryl, andpolyalkalene (e.g., methoxy ethoxy ethoxy, ethoxy ethoxy, and—(OCH₂CH₂)_(n)OH, n=1-50), phenyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted phenyl, pyridyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted pyridyl, bipyridyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted bipyridyltripyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedtripyridyl, furyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted furyl, thienyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted thienyl, pyrrolyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrrolyl, pyrazolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrazolyl,oxazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedoxazolyl, thiazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted thiazolyl, imidazolyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted imidazolyl, pyrazinyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrazinyl,benzooxadizolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted benzooxadizolyl, benzothiadizolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbenzothiadizolyl, fluorenyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted fluorenyl, triphenylaminyl-substitutedfluorenyl, diphenylaminyl-substituted fluorenyl, alkyl-substitutedcarbazolyl, alkyl-substituted triphenylaminyl and alkyl-substitutedthiophenyl. As exemplary embodiments, alkyl substituted phenyl caninclude 2-alkylphenyl, 3-alkylphenyl, 4-alkylphenyl, 2,4-dialkylphenyl,3,5-dialkylphenyl, 3,4-dialkylphenyl; alkyl-substituted fluorenyl caninclude 9, 9-dialkyl-substituted fluorenyl,7-alkyl-9,9-dialkyl-substituted fluorenyl,6-alkyl-9,9-dialkyl-substituted fluorenyl,7-triphenylaminyl-9,9-dialkyl-substituted fluorenyl and7-diphenylaminyl-9,9-dialkyl-substituted fluorenyl; alkyl-substitutedcarbazolyl can include N-alkyl-substituted carbazolyl,6-alkyl-substituted carbazolyl and 7-alkyl-substituted carbazolyl;alkyl-substituted triphenylaminyl can include 4′-alkyl-substitutedtriphenylaminyl, 3′-alkyl-substituted triphenylaminyl,3′,4′-dialkyl-substituted triphenylaminyl and 4′,4″-alkyl-substitutedtriphenylaminyl; alkyl-substituted thiophenyl can include2-alkylthiophenyl, 3-alkylthiophenyl, and 4-alkylthiophenyl,N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, and N-dialkoxyphenyl-4-phenyl.The narrow-band monomer can be integrated into a backbone of the polymer(e.g., polymerized in the polymer) and/or covalently attached to thebackbone, a terminus, or a sidechain of the polymer through at least oneattachment, e.g., to R¹, R^(2A), R^(2B), or a combination thereof. Theparentheses indicate points of attachment of the monomer to the backboneof the polymer.

In some embodiments, the hybrid polymer dots of the present disclosureinclude a polymer that includes a narrow-band monomer having thestructure of Formula (IV):

wherein each of R¹, R^(2A), R^(2B), R^(3A), and R^(3B) is independentlyselected from the group consisting of, but not limited to, hydrogen (H),deuterium (D), halogen, direct or branched alkyl, heteroalkyl,heterocycloalkyl, heterocycloalkylene, alkoxy, aryl, hydroxyl, cyano,nitro, ether and its derivatives, ester and its derivatives, alkylketone, alkylester, arylester, alkynyl, alkyl amine, fluoroalkyl,fluoroaryl, and polyalkalene (e.g., methoxy ethoxy ethoxy, ethoxyethoxy, and —(OCH₂CH₂)_(n)OH, n=1-50), phenyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted phenyl, pyridyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted pyridyl, bipyridyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted bipyridyltripyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedtripyridyl, furyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted furyl, thienyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted thienyl, pyrrolyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrrolyl, pyrazolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrazolyl,oxazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedoxazolyl, thiazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted thiazolyl, imidazolyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted imidazolyl, pyrazinyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrazinyl,benzooxadizolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted benzooxadizolyl, benzothiadizolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbenzothiadizolyl, fluorenyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted fluorenyl, triphenylaminyl-substitutedfluorenyl, diphenylaminyl-substituted fluorenyl, alkyl-substitutedcarbazolyl, alkyl-substituted triphenylaminyl and alkyl-substitutedthiophenyl. As exemplary embodiments, alkyl substituted phenyl caninclude 2-alkylphenyl, 3-alkylphenyl, 4-alkylphenyl, 2,4-dialkylphenyl,3,5-dialkylphenyl, 3,4-dialkylphenyl; alkyl-substituted fluorenyl caninclude 9, 9-dialkyl-substituted fluorenyl,7-alkyl-9,9-dialkyl-substituted fluorenyl,6-alkyl-9,9-dialkyl-substituted fluorenyl,7-triphenylaminyl-9,9-dialkyl-substituted fluorenyl and7-diphenylaminyl-9,9-dialkyl-substituted fluorenyl; alkyl-substitutedcarbazolyl can include N-alkyl-substituted carbazolyl,6-alkyl-substituted carbazolyl and 7-alkyl-substituted carbazolyl;alkyl-substituted triphenylaminyl can include 4′-alkyl-substitutedtriphenylaminyl, 3′-alkyl-substituted triphenylaminyl,3′,4′-dialkyl-substituted triphenylaminyl and 4′,4″-alkyl-substitutedtriphenylaminyl; alkyl-substituted thiophenyl can include2-alkylthiophenyl, 3-alkylthiophenyl, and 4-alkylthiophenyl,N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, and N-dialkoxyphenyl-4-phenyl.The narrow-band monomer can be integrated into a backbone of the polymer(e.g., polymerized in the polymer) and/or covalently attached to thebackbone, a terminus, or a sidechain of the polymer through at least oneattachment to R¹, R^(2A), R^(2B), R^(3A), and R^(3B) or a combinationthereof.

In some embodiments, the hybrid polymer dots of the present disclosureinclude a polymer that includes a narrow-band monomer having thestructure of Formula (V):

wherein each of R¹, R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B),R^(5A), and R^(5B) is independently selected from the group consistingof, but not limited to, hydrogen (H), deuterium (D), halogen, direct orbranched alkyl, heteroalkyl, heterocycloalkyl, heterocycloalkylene,alkoxy, aryl, hydroxyl, cyano, nitro, ether and its derivatives, esterand its derivatives, alkyl ketone, alkylester, arylester, alkynyl, alkylamine, fluoroalkyl, fluoroaryl, and polyalkalene (e.g., methoxy ethoxyethoxy, ethoxyethoxy, and —(OCH₂CH₂)_(n)OH, n=1-50), phenyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted phenyl,pyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyridyl, bipyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted bipyridyl tripyridyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted tripyridyl, furyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted furyl, thienyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted thienyl,pyrrolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrrolyl, pyrazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted pyrazolyl, oxazolyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted oxazolyl, thiazolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted thiazolyl,imidazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedimidazolyl, pyrazinyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted pyrazinyl, benzooxadizolyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted benzooxadizolyl,benzothiadizolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted benzothiadizolyl, fluorenyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted fluorenyl,triphenylaminyl-substituted fluorenyl, diphenylaminyl-substitutedfluorenyl, alkyl-substituted carbazolyl, alkyl-substitutedtriphenylaminyl and alkyl-substituted thiophenyl. As exemplaryembodiments, alkyl substituted phenyl can include 2-alkylphenyl,3-alkylphenyl, 4-alkylphenyl, 2,4-dialkylphenyl, 3,5-dialkylphenyl,3,4-dialkylphenyl; alkyl-substituted fluorenyl can include 9,9-dialkyl-substituted fluorenyl, 7-alkyl-9,9-dialkyl-substitutedfluorenyl, 6-alkyl-9,9-dialkyl-substituted fluorenyl,7-triphenylaminyl-9,9-dialkyl-substituted fluorenyl and7-diphenylaminyl-9,9-dialkyl-substituted fluorenyl; alkyl-substitutedcarbazolyl can include N-alkyl-substituted carbazolyl,6-alkyl-substituted carbazolyl and 7-alkyl-substituted carbazolyl;alkyl-substituted triphenylaminyl can include 4′-alkyl-substitutedtriphenylaminyl, 3′-alkyl-substituted triphenylaminyl,3′,4′-dialkyl-substituted triphenylaminyl and 4′,4″-alkyl-substitutedtriphenylaminyl; alkyl-substituted thiophenyl can include2-alkylthiophenyl, 3-alkylthiophenyl, and 4-alkylthiophenyl,N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, and N-dialkoxyphenyl-4-phenyl.The narrow-band monomer can be integrated into a backbone of the polymer(e.g., copolymerized in the polymer) and/or covalently attached to thebackbone, a terminus, or a sidechain of the polymer through at least oneattachment to R¹, R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B),R^(5A), R^(5B), or a combination thereof. In certain embodiments, thenarrow-band monomers can be integrated into the backbone by attachmentto the R^(5A) and R^(5B) groups.

In some embodiments, the hybrid polymer dots of the present disclosureinclude a polymer that includes a narrow-band monomer having thestructure of Formula (VI):

wherein each of R^(1A), R^(1B), R^(2A), R^(2B), R^(3A) and R^(3B) isindependently selected from the group consisting of, but not limited to,hydrogen (H), deuterium (D), halogen, direct or branched alkyl,heteroalkyl, heterocycloalkyl, heterocycloalkylene, alkoxy, aryl,hydroxyl, cyano, nitro, ether and its derivatives, ester and itsderivatives, alkyl ketone, alkylester, arylester, alkynyl, alkyl amine,fluoroalkyl, fluoroaryl, and polyalkalene (e.g., methoxyethoxyethoxy,ethoxyethoxy, and —(OCH₂CH₂)_(n)OH, n=1-50), phenyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted phenyl, pyridyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyridyl,bipyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbipyridyl tripyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted tripyridyl, furyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted furyl, thienyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted thienyl, pyrrolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrrolyl,pyrazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazolyl, oxazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted oxazolyl, thiazolyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted thiazolyl, imidazolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted imidazolyl,pyrazinyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazinyl, benzooxadizolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted benzooxadizolyl, benzothiadizolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbenzothiadizolyl, fluorenyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted fluorenyl, triphenylaminyl-substitutedfluorenyl, diphenylaminyl-substituted fluorenyl, alkyl-substitutedcarbazolyl, alkyl-substituted triphenylaminyl and alkyl-substitutedthiophenyl. As exemplary embodiments, alkyl substituted phenyl caninclude 2-alkylphenyl, 3-alkylphenyl, 4-alkylphenyl, 2,4-dialkylphenyl,3,5-dialkylphenyl, 3,4-dialkylphenyl; alkyl-substituted fluorenyl caninclude 9, 9-dialkyl-substituted fluorenyl,7-alkyl-9,9-dialkyl-substituted fluorenyl,6-alkyl-9,9-dialkyl-substituted fluorenyl,7-triphenylaminyl-9,9-dialkyl-substituted fluorenyl and7-diphenylaminyl-9,9-dialkyl-substituted fluorenyl; alkyl-substitutedcarbazolyl can include N-alkyl-substituted carbazolyl,6-alkyl-substituted carbazolyl and 7-alkyl-substituted carbazolyl;alkyl-substituted triphenylaminyl can include 4′-alkyl-substitutedtriphenylaminyl, 3′-alkyl-substituted triphenylaminyl,3′,4′-dialkyl-substituted triphenylaminyl and 4′,4″-alkyl-substitutedtriphenylaminyl; alkyl-substituted thiophenyl can include2-alkylthiophenyl, 3-alkylthiophenyl, and 4-alkylthiophenyl,N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, and N-dialkoxyphenyl-4-phenyl.The narrow-band monomer can be integrated into a backbone of the polymer(e.g., polymerized in the polymer) and/or covalently attached to thebackbone, a terminus, or a sidechain of the polymer through at least oneattachment to R^(1A), R^(1B), R^(2A), R^(2B), R^(3A), R^(3B), or acombination thereof.

In some embodiments, the hybrid polymer dots of the present disclosureinclude a polymer that includes a narrow-band monomer having thestructure of Formula (VII):

wherein each of R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A)and R^(5B) is independently selected from the group consisting of, butnot limited to, hydrogen (H), deuterium (D), halogen, direct or branchedalkyl, heteroalkyl, heterocycloalkyl, heterocycloalkylene, alkoxy, aryl,hydroxyl, cyano, nitro, ether and its derivatives, ester and itsderivatives, alkyl ketone, alkylester, arylester, alkynyl, alkyl amine,fluoroalkyl, fluoroaryl, and polyalkalene (e.g., methoxy ethoxy ethoxy,ethoxyethoxy, and —(OCH₂CH₂)_(n)OH, n=1-50), phenyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted phenyl, pyridyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyridyl,bipyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbipyridyl tripyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted tripyridyl, furyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted furyl, thienyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted thienyl, pyrrolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrrolyl,pyrazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazolyl, oxazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted oxazolyl, thiazolyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted thiazolyl, imidazolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted imidazolyl,pyrazinyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazinyl, benzooxadizolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted benzooxadizolyl, benzothiadizolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbenzothiadizolyl, fluorenyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted fluorenyl, triphenylaminyl-substitutedfluorenyl, diphenylaminyl-substituted fluorenyl, alkyl-substitutedcarbazolyl, alkyl-substituted triphenylaminyl and alkyl-substitutedthiophenyl. As exemplary embodiments, alkyl substituted phenyl caninclude 2-alkylphenyl, 3-alkylphenyl, 4-alkylphenyl, 2,4-dialkylphenyl,3,5-dialkylphenyl, 3,4-dialkylphenyl; alkyl-substituted fluorenyl caninclude 9, 9-dialkyl-substituted fluorenyl,7-alkyl-9,9-dialkyl-substituted fluorenyl,6-alkyl-9,9-dialkyl-substituted fluorenyl,7-triphenylaminyl-9,9-dialkyl-substituted fluorenyl and7-diphenylaminyl-9,9-dialkyl-substituted fluorenyl; alkyl-substitutedcarbazolyl can include N-alkyl-substituted carbazolyl,6-alkyl-substituted carbazolyl and 7-alkyl-substituted carbazolyl;alkyl-substituted triphenylaminyl can include 4′-alkyl-substitutedtriphenylaminyl, 3′-alkyl-substituted triphenylaminyl,3′,4′-dialkyl-substituted triphenylaminyl and 4′,4″-alkyl-substitutedtriphenylaminyl; alkyl-substituted thiophenyl can include2-alkylthiophenyl, 3-alkylthiophenyl, and 4-alkylthiophenyl,N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, and N-dialkoxyphenyl-4-phenylThe narrow-band monomer can be integrated into a backbone of the polymer(e.g., polymerized in the polymer) and/or covalently attached to thebackbone, a terminus, or a sidechain of the polymer) through at leastone attachment to R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B),R^(5A), R^(5B), or a combination thereof.

In some embodiments, the hybrid polymer dots of the present disclosureinclude a polymer that includes a narrow-band monomer having thestructure of Formula (VIII):

wherein each of R¹, R^(2A), R^(2B), R^(3A), R^(3B), R^(4A) and R^(4B),is independently selected from the group consisting of, but not limitedto, hydrogen (H), deuterium (D), halogen, direct or branched alkyl,heteroalkyl, heterocycloalkyl, heterocycloalkylene, alkoxy, aryl,hydroxyl, cyano, nitro, ether and its derivatives, ester and itsderivatives, alkyl ketone, alkylester, arylester, alkynyl, alkyl amine,fluoroalkyl, fluoroaryl, and polyalkalene (e.g., methoxy ethoxy ethoxy,ethoxy ethoxy, and —(OCH₂CH₂)_(n)OH, n=1-50), phenyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted phenyl, pyridyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyridyl,bipyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbipyridyl tripyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted tripyridyl, furyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted furyl, thienyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted thienyl, pyrrolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrrolyl,pyrazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazolyl, oxazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted oxazolyl, thiazolyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted thiazolyl, imidazolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted imidazolyl,pyrazinyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazinyl, benzooxadizolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted benzooxadizolyl, benzothiadizolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbenzothiadizolyl, fluorenyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted fluorenyl, triphenylaminyl-substitutedfluorenyl, diphenylaminyl-substituted fluorenyl, alkyl-substitutedcarbazolyl, alkyl-substituted triphenylaminyl and alkyl-substitutedthiophenyl. As exemplary embodiments, alkyl substituted phenyl caninclude 2-alkylphenyl, 3-alkylphenyl, 4-alkylphenyl, 2,4-dialkylphenyl,3,5-dialkylphenyl, 3,4-dialkylphenyl; alkyl-substituted fluorenyl caninclude 9, 9-dialkyl-substituted fluorenyl,7-alkyl-9,9-dialkyl-substituted fluorenyl,6-alkyl-9,9-dialkyl-substituted fluorenyl,7-triphenylaminyl-9,9-dialkyl-substituted fluorenyl and7-diphenylaminyl-9,9-dialkyl-substituted fluorenyl; alkyl-substitutedcarbazolyl can include N-alkyl-substituted carbazolyl,6-alkyl-substituted carbazolyl and 7-alkyl-substituted carbazolyl;alkyl-substituted triphenylaminyl can include 4′-alkyl-substitutedtriphenylaminyl, 3′-alkyl-substituted triphenylaminyl,3′,4′-dialkyl-substituted triphenylaminyl and 4′,4″-alkyl-substitutedtriphenylaminyl; alkyl-substituted thiophenyl can include2-alkylthiophenyl, 3-alkylthiophenyl, and 4-alkylthiophenyl,N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, and N-dialkoxyphenyl-4-phenyl,and wherein each of R^(5A), R^(5B), R^(6A) and R^(6B) are independentlyselected from the group consisting of, but not limited to, hydrogen (H),deuterium (D), halogen, direct or branched alkyl, heteroalkyl,heterocycloalkyl, heterocycloalkylene, alkoxy, aryl, hydroxyl, cyano,nitro, ether and its derivatives, ester and its derivatives, alkylketone, alkylester, arylester, alkynyl, alkyl amine, fluoroalkyl,fluoroaryl, and polyalkalene (e.g., methoxy ethoxy ethoxy, ethoxyethoxy, and —(OCH₂CH₂)_(n)OH, n=1-50), phenyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted phenyl, pyridyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted pyridyl, bipyridyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted bipyridyltripyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedtripyridyl, furyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted furyl, thienyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted thienyl, pyrrolyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrrolyl, pyrazolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrazolyl,oxazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedoxazolyl, thiazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted thiazolyl, imidazolyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted imidazolyl, pyrazinyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrazinyl,benzooxadizolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted benzooxadizolyl, benzothiadizolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbenzothiadizolyl, fluorenyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted fluorenyl, triphenylaminyl-substitutedfluorenyl, diphenylaminyl-substituted fluorenyl, alkyl-substitutedcarbazolyl, alkyl-substituted triphenylaminyl and alkyl-substitutedthiophenyl. As exemplary embodiments, alkyl substituted phenyl caninclude 2-alkylphenyl, 3-alkylphenyl, 4-alkylphenyl, 2,4-dialkylphenyl,3,5-dialkylphenyl, 3,4-dialkylphenyl; alkyl-substituted fluorenyl caninclude 9, 9-dialkyl-substituted fluorenyl,7-alkyl-9,9-dialkyl-substituted fluorenyl,6-alkyl-9,9-dialkyl-substituted fluorenyl,7-triphenylaminyl-9,9-dialkyl-substituted fluorenyl and7-diphenylaminyl-9,9-dialkyl-substituted fluorenyl; alkyl-substitutedcarbazolyl can include N-alkyl-substituted carbazolyl,6-alkyl-substituted carbazolyl and 7-alkyl-substituted carbazolyl;alkyl-substituted triphenylaminyl can include 4′-alkyl-substitutedtriphenylaminyl, 3′-alkyl-substituted triphenylaminyl,3′,4′-dialkyl-substituted triphenylaminyl and 4′,4″-alkyl-substitutedtriphenylaminyl; alkyl-substituted thiophenyl can include2-alkylthiophenyl, 3-alkylthiophenyl, and 4-alkylthiophenyl,N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, and N-dialkoxyphenyl-4-phenyl.The narrow-band monomer can be integrated into a backbone of the polymer(e.g., copolymerized in the polymer) and/or covalently attached to thebackbone, a terminus, or a sidechain of the polymer) through at leastone attachment to R¹, R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B),R^(5A), R^(5B), R^(6A), R^(6B) or a combination thereof.

In some embodiments, the hybrid polymer dots of the present disclosureinclude a polymer that includes a narrow-band monomer having thestructure of Formula (IX):

wherein X has the structure of any one of Formulae (X), (XI), (XII), and(XIII) or their derivatives:

and wherein each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²,R¹³, R¹⁴ and R¹⁵ in Formulae (X), (XI), (XII), and (XIII) isindependently selected from the group consisting of, but not limited to,hydrogen (H), deuterium (D), halogen, direct or branched alkyl,heteroalkyl, heterocycloalkyl, heterocycloalkylene, alkoxy, aryl,hydroxyl, cyano, nitro, ether and its derivatives, ester and itsderivatives, alkyl ketone, alkylester, arylester, alkynyl, alkyl amine,fluoroalkyl, fluoroaryl, and polyalkalene (e.g., methoxyethoxyethoxy,ethoxy ethoxy, and —(OCH₂CH₂)_(n)OH, n=1-50), phenyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted phenyl, pyridyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyridyl,bipyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbipyridyl tripyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted tripyridyl, furyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted furyl, thienyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted thienyl, pyrrolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrrolyl,pyrazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazolyl, oxazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted oxazolyl, thiazolyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted thiazolyl, imidazolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted imidazolyl,pyrazinyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazinyl, benzooxadizolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted benzooxadizolyl, benzothiadizolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbenzothiadizolyl, fluorenyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted fluorenyl, triphenylaminyl-substitutedfluorenyl, diphenylaminyl-substituted fluorenyl, alkyl-substitutedcarbazolyl, alkyl-substituted triphenylaminyl and alkyl-substitutedthiophenyl. As exemplary embodiments, alkyl substituted phenyl caninclude 2-alkylphenyl, 3-alkylphenyl, 4-alkylphenyl, 2,4-dialkylphenyl,3,5-dialkylphenyl, 3,4-dialkylphenyl; alkyl-substituted fluorenyl caninclude 9, 9-dialkyl-substituted fluorenyl,7-alkyl-9,9-dialkyl-substituted fluorenyl,6-alkyl-9,9-dialkyl-substituted fluorenyl,7-triphenylaminyl-9,9-dialkyl-substituted fluorenyl and7-diphenylaminyl-9,9-dialkyl-substituted fluorenyl; alkyl-substitutedcarbazolyl can include N-alkyl-substituted carbazolyl,6-alkyl-substituted carbazolyl and 7-alkyl-substituted carbazolyl;alkyl-substituted triphenylaminyl can include 4′-alkyl-substitutedtriphenylaminyl, 3′-alkyl-substituted triphenylaminyl,3′,4′-dialkyl-substituted triphenylaminyl and 4′,4″-alkyl-substitutedtriphenylaminyl; alkyl-substituted thiophenyl can include2-alkylthiophenyl, 3-alkylthiophenyl, and 4-alkylthiophenyl,N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, and N-dialkoxyphenyl-4-phenyl.When X represents naphthalene and its derivatives, the narrow-bandmonomer can be integrated into a backbone (e.g., polymerized in thepolymer) and/or covalently attached to the backbone, a terminus, or asidechain of the polymer) of the polymer through at least one attachmentto R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³ or a combination thereof. When Xrepresents anthracene and its derivatives, the narrow-band monomer canbe integrated into a backbone of the polymer and/or covalently attachedto the backbone, a terminus, or a sidechain of the polymer through atleast one attachment to R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵ or acombination thereof.

Narrow band monomers of the present disclosure can further includedipyrrin derivatives. Dipyrrin and dipyrrin derivatives can bepolymerized to form polymers (e.g., homopolymers or heteropolymers)and/or can be attached (e.g., covalently attached) to a polymerbackbone, sidechain and/or terminus. For example, the hybrid polymerdots of the present disclosure can include a polymer that includes anarrow-band monomer having the structure of Formula (XIV):

wherein M is a metal. Examples of M can be, but is not limited to, Na,Li, Zn, Co, or Si. X can include substituents such as, but not limitedto, halogen, alkyl, phenyl, alkylphenyl, thiophenyl, alkylthiophenyl,alkoxyl, alkoxylphenyl, alkylthiophenyl, ester, or hydroxyl. The numberof X groups (n) can be 1 or more than 1, and n can be 0, 1, 2, 3, 4.Each of R¹, R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), and R^(4B) can beindependently selected from the group consisting of, but not limited to,hydrogen (H), deuterium (D), halogen, direct or branched alkyl,heteroalkyl, heterocycloalkyl, heterocycloalkylene, alkoxy, aryl,hydroxyl, cyano, nitro, ether and its derivatives, ester and itsderivatives, alkyl ketone, alkylester, arylester, alkynyl, alkyl amine,fluoroalkyl, fluoroaryl, and polyalkalene (e.g., methoxyethoxyethoxy,ethoxy ethoxy, and —(OCH₂CH₂)_(n)OH, n=1-50), phenyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted phenyl, pyridyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyridyl,bipyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbipyridyl tripyridyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted tripyridyl, furyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted furyl, thienyl, alkyl-(alkoxy-,aryl-, fluoroalkyl-, fluoroaryl-)substituted thienyl, pyrrolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted pyrrolyl,pyrazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazolyl, oxazolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted oxazolyl, thiazolyl, alkyl-(alkoxy-, aryl-,fluoroalkyl-, fluoroaryl-)substituted thiazolyl, imidazolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substituted imidazolyl,pyrazinyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedpyrazinyl, benzooxadizolyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted benzooxadizolyl, benzothiadizolyl,alkyl-(alkoxy-, aryl-, fluoroalkyl-, fluoroaryl-)substitutedbenzothiadizolyl, fluorenyl, alkyl-(alkoxy-, aryl-, fluoroalkyl-,fluoroaryl-)substituted fluorenyl, triphenylaminyl-substitutedfluorenyl, diphenylaminyl-substituted fluorenyl, alkyl-substitutedcarbazolyl, alkyl-substituted triphenylaminyl and alkyl-substitutedthiophenyl. As exemplary embodiments, alkyl substituted phenyl caninclude 2-alkylphenyl, 3-alkylphenyl, 4-alkylphenyl, 2,4-dialkylphenyl,3,5-dialkylphenyl, 3,4-dialkylphenyl; alkyl-substituted fluorenyl caninclude 9, 9-dialkyl-substituted fluorenyl,7-alkyl-9,9-dialkyl-substituted fluorenyl,6-alkyl-9,9-dialkyl-substituted fluorenyl,7-triphenylaminyl-9,9-dialkyl-substituted fluorenyl and7-diphenylaminyl-9,9-dialkyl-substituted fluorenyl; alkyl-substitutedcarbazolyl can include N-alkyl-substituted carbazolyl,6-alkyl-substituted carbazolyl and 7-alkyl-substituted carbazolyl;alkyl-substituted triphenylaminyl can include 4′-alkyl-substitutedtriphenylaminyl, 3′-alkyl-substituted triphenylaminyl,3′,4′-dialkyl-substituted triphenylaminyl and 4′,4″-alkyl-substitutedtriphenylaminyl; alkyl-substituted thiophenyl can include2-alkylthiophenyl, 3-alkylthiophenyl, and 4-alkylthiophenyl,N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, and N-dialkoxyphenyl-4-phenyl.The narrow-band monomer can be integrated into a backbone of the polymer(e.g., polymerized in the polymer) and/or covalently attached to thebackbone, a terminus, or a sidechain of the polymer) through at leastone attachment to R¹, R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), ora combination thereof.

In some embodiments, the narrow-band emissive polymers for making hybridpolymer dots include squaraine and squaraine derivatives as narrow-bandmonomers. Squaraine derivatives include but are not limited to theiralkyl derivatives, aryl derivatives, alkyne derivatives, aromaticderivatives, alkoxide derivatives, aza derivatives, their extendedsystems and analogues. The narrow-band emissive polymers can alsoinclude any other monomers. The squaraine and their derivatives can beenergy acceptors and other monomers can be energy donors so that thefinal hybrid polymer dots can exhibit narrow-band emissions. Thenarrow-band emissive chromophoric polymers in good solvents may exhibitbroad emissions or narrow emissions. However, in some embodiments, theirnanoparticle form gives narrow-band emissions. In some embodiments, theemission full width at half maxium (FWHM) of the above hybrid polymerdots is less than 70 nm. In certain embodiments, the FWHM is less than60 nm, less than 50 nm, less than 40 nm, less than 30 nm, or less than20 nm.

Suitable squaraine derivatives for use in the present disclosure caninclude the following structures described below. Squaraine andsquaraine derivatives can be polymerized to form polymers (e.g.,homopolymers or heteropolymers) and/or can be attached (e.g., covalentlyattached) to a polymer backbone, sidechain and/or terminus. Hybridpolymer dots of the present disclosure can include a polymer thatincludes a narrow-band monomer having the structure of Formula (XV):

wherein each of X¹ and X² is independently selected from the groupconsisting of oxygen, sulfur and nitrogen; each of R^(1A) and R^(1B) isindependently selected from the group consisting of, but not limited to,alkylene, alkenylene, arylene, heteroarylene, phenylene, azulene,cycloalkylene, and heterocycloalkylene; and each of R^(2A) and R^(2B) isa reactive group independently selected from the group consisting of,but not limited to, a halide, hydroxyl, and amino. Other reactive groupscan be used. In some embodiments, the halide is a chloro, a bromo, or aniodo group. The reactive group can be used to integrate the monomer intoa polymer, e.g., along the backbone of the polymer (e.g., bypolymerizing in the polymer) and/or to attach the monomer by covalentattachment to the backbone, a terminus, or a sidechain of the polymer.The narrow-band monomer can be integrated into a backbone of the polymer(e.g., polymerized in the polymer) and/or covalently attached to thebackbone, a terminus, or a sidechain of the polymer) through at leastone attachment to R^(1A), R^(1B), R^(2A), R^(2B), or a combinationthereof.

The present disclosure can include oxygen-containing squarainederivatives. Hybrid polymer dots of the present disclosure can include apolymer that includes a narrow-band monomer having the structure ofFormula (XVI):

wherein each of R^(1A) and R^(1B) is independently selected from thegroup consisting of, but not limited to, alkylene, alkenylene, arylene,heteroarylene, phenylene, azulene, cycloalkylene, andheterocycloalkylene; and each of R^(2A) and R^(2B) is a reactive groupindependently selected from the group consisting of, but not limited to,a halide, hydroxyl, and amino. Other reactive groups can be used. Insome embodiments, the halide is a chloro, a bromo, or an iodo group. Thereactive group can be used to integrate the monomer into a polymer(e.g., along the backbone of the polymer by polymerizing in the polymer)and/or to attach the monomer by covalent attachment to the backbone, aterminus, or a sidechain of the polymer.

Hybrid polymer dots of the present disclosure can include a polymer thatincludes a narrow-band monomer having the structure of Formula (XVII):

wherein each of R^(1A) and R^(1B) is independently selected from thegroup consisting of, but not limited to, hydrogen, methyl, alkyl,phenyl, araalkyl, and alkoxy-phenyl; each of R^(2A) and R^(2B) isindependently selected from the group consisting of, but not limited to,hydrogen, methyl, alkyl, phenyl, araalkyl, and alkoxy-phenyl; each ofR^(3A) and R^(3B) is a reactive group independently selected from thegroup consisting of, but not limited to, chloro, bromo, iodo, andhydroxyl; each of R^(4A) and R^(4B) is independently is selected from agroup consisting of, but not limited to, hydroxyl, hydrogen, alkyl,phenyl, araalkyl, and alkoxy-phenyl; and each of R^(5A) and R^(5B) isindependently selected from the group consisting of, but not limited to,hydrogen, methyl, alkyl, phenyl, araalkyl, and alkoxy-phenyl. Otherreactive groups can be used. The reactive group can be used to integratethe monomer into a polymer (e.g., along the backbone of the polymer bypolymerizing in the polymer) and/or to attach the monomer by covalentattachment to the backbone, a terminus, or a sidechain of the polymer.

Hybrid polymer dots of the present disclosure can include a polymer thatincludes a narrow-band monomer having the structure of Formula (XVIII):

wherein each of R^(1A) and R^(1B) is a reactive group independentlyselected from the group consisting of, but not limited to, chloro,bromo, iodo, and hydroxyl; and each of R^(2A) and R^(2B) is selectedfrom the group consisting of, but not limited to, hydrogen, methyl,alkyl, phenyl, araalkyl, and alkoxy-phenyl. Other reactive groups can beused. The reactive group can be used to integrate the monomer into apolymer (e.g., along the backbone of the polymer by polymerizing in thepolymer) and/or attach the monomer by covalent attachment to thebackbone, a terminus, or a sidechain of the polymer.

Hybrid polymer dots of the present disclosure can include a polymer thatincludes a narrow-band monomer having the structure of Formula (XIX):

wherein each of X¹ and X² is independently selected from the groupconsisting of carbon, sulfur, nitrogen, and selenium; each of R^(1A) andR^(1B) is a reactive group independently selected from the groupconsisting of, but not limited to, chloro, bromo, iodo, and hydroxyl;and each of R^(2A) and R^(2B) is independently selected from the groupconsisting of, but not limited to, hydrogen, methyl, alkyl, phenyl,araalkyl, alkoxy-phenyl, N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, andN-dialkoxylphenyl-4-phenyl. Other reactive groups can be used. Thereactive group can be used to integrate the monomer into a polymer(e.g., along the backbone of the polymer by polymerizing in the polymer)and/or attach the monomer by covalent attachment to the backbone, aterminus, or a sidechain of the polymer.

Hybrid polymer dots of the present disclosure can include a polymer thatincludes a narrow-band monomer having the structure of Formula (XX):

wherein each of R^(2A) and R^(2B) is a reactive group independentlyselected from the group consisting of, but not limited to, chloro,bromo, iodo, and hydroxyl; and each of R^(1A) and R^(1B) is selectedfrom the group consisting of, but not limited to, hydrogen, methyl,alkyl, phenyl, araalkyl, alkoxy-phenyl, N-dialkyl-4-phenyl,N-diphenyl-4-phenyl, and N-dialkoxylphenyl-4-phenyl. Other reactivegroups can be used. The reactive group can be used to integrate themonomer into a polymer (e.g., along the backbone of the polymer bypolymerizing in the polymer) and/or attach the monomer by covalentattachment to the backbone, a terminus, or a sidechain of the polymer.

The present disclosure can include sulfur-containing squarainederivatives. Hybrid polymer dots of the present disclosure can include apolymer that includes a narrow-band monomer having the structure ofFormula (XXI):

wherein each of R^(1A) and R^(1B) is independently selected from thegroup consisting of, but not limited to, alkylene, alkenylene, arylene,heteroarylene, phenylene, azulene, cycloalkylene, andheterocycloalkylene; and each of R^(2A) and R^(2B) is a reactive groupindependently selected from the group consisting of, but not limited to,a halide, hydroxyl, and amino. In some embodiments, the halide is achloro, a bromo, or an iodo group. Other reactive groups can be used.The reactive group can be used to integrate the monomer into a polymer(e.g., along the backbone of the polymer by polymerizing in the polymer)and/or attach the monomer by covalent attachment to the backbone, aterminus, or a sidechain of the polymer.

Hybrid polymer dots of the present disclosure can include a polymer thatincludes a narrow-band monomer having the structure of Formula (XXII):

wherein each of X¹ and X² is independently selected from the groupconsisting of carbon, sulfur, nitrogen, and selenium; each of R^(1A) andR^(1B) is a reactive group independently selected from the groupconsisting of, but not limited to, chloro, bromo, iodo, and hydroxyl;and each of R^(2A) and R^(2B) is independently selected from the groupconsisting of, but not limited to, hydrogen, methyl, alkyl, phenyl,araalkyl, alkoxy-phenyl, N-dialkyl-4-phenyl, N-diphenyl-4-phenyl, andN-dialkoxylphenyl-4-phenyl. Other reactive groups can be used. In someembodiments, the halide is a chloro, a bromo, or an iodo group. Thereactive group can be used to integrate the monomer into a polymer(e.g., along the backbone of the polymer by polymerizing in the polymer)and/or attach the monomer by covalent attachment to the backbone, aterminus, or a sidechain of the polymer.

The present disclosure can include nitrogen-containing squarainederivatives. Hybrid polymer dots of the present disclosure can include apolymer that includes a narrow-band monomer having the structure ofFormula (XXIII):

wherein each of R^(1A) and R^(1B) is independently selected from thegroup consisting of, but not limited to, alkylene, alkenylene, arylene,heteroarylene, phenylene, azulene, cycloalkylene, andheterocycloalkylene; each of R^(2A) and R^(2B) is a reactive groupindependently selected from the group consisting of, but not limited to,a halide, hydroxyl, and amino; and each of R^(3A) and R^(3B) isindependently selected from the group consisting of hydrogen, methyl,alkyl, phenyl, aralkyl, and alkoxy-phenyl. Other reactive groups can beused. In some embodiments, the halide is a chloro, a bromo, or an iodogroup. The reactive group can be used to integrate the monomer alonginto a polymer (e.g., along the backbone of the polymer by polymerizingin the polymer) and/or attach the monomer by covalent attachment to thebackbone, a terminus, or a sidechain of the polymer.

Hybrid polymer dots of the present disclosure can include a polymer thatincludes a narrow-band monomer having the structure of Formula (XXIV):

wherein each of R^(1A), R^(1B), R^(2A) and R^(2B) are independentlyselected from the group consisting of, but not limited to, hydrogen,deuterium, alkyl, aryl, acetyl, and hydroxyl; and each of R^(3A),R^(3B), R^(4A) and R^(4B) are independently selected from the groupconsisting of, but not limited to, hydrogen, deuterium, alkyl, aryl,amino, sulfide, aldehyde, ester, ether, carboxyl, hydroxyl, and halide.The narrow-band monomer can be integrated into a backbone of the polymer(e.g., along the backbone of the polymer by polymerizing in the polymer)and/or attached by covalent attachment to the backbone, a terminus, or asidechain of the polymer through at least one attachment to R^(1A),R^(1B), R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B) or a combinationthereof.

Hybrid polymer dots of the present disclosure can include a polymer thatincludes a narrow-band monomer having the structure of Formula (XXV):

wherein each of R^(1A), R^(1B), R^(2A) and R^(2B) are independentlyselected from the group consisting of, but not limited to, hydrogen,deuterium, alkyl, aryl, acetyl, and hydroxyl; and each of R^(3A),R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(6A) and R^(6B) areindependently selected from the group consisting of, but not limited to,hydrogen, deuterium, alkyl, aryl, amino, sulfide, aldehyde, ester,ether, carboxyl, hydroxyl, and halide. The narrow-band monomer can beintegrated into a backbone of the polymer (e.g., along the backbone ofthe polymer by polymerizing in the polymer) and/or attached by covalentattachment to the backbone, a terminus, or a sidechain of the polymerthrough at least one attachment to R^(1A), R^(1B), R^(2A), R^(2B),R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(6A), R^(6B) or acombination thereof.

Hybrid polymer dots of the present disclosure can include a polymer thatincludes a narrow-band monomer having the structure of Formula (XXVI):

wherein each of R^(1A), R^(1B), R^(1C), R^(2A), R^(2B), R^(2C), R^(3A),R^(3B), R^(3C), R^(4A), R^(4B), R^(4C), R^(5A), R^(5B), R^(5C), R^(6A),R^(6B), and R^(6C) are independently selected from the group consistingof, but not limited to, hydrogen, deuterium, alkyl, aryl, amino,sulfide, aldehyde, ester, ether, carboxyl, hydroxyl, and halide, andeach of R^(7A), R^(7B), and R^(7C) is independently selected from thegroup consisting of, but not limited to, hydrogen, deuterium, alkyl,aryl and acetyl. The narrow-band monomer can be integrated into abackbone of the polymer (e.g., along the backbone of the polymer bypolymerizing in the polymer) and/or attached by covalent attachment tothe backbone, a terminus, or a sidechain of the polymer through at leastone attachment to R^(1A), R^(1B), R^(1C), R^(2A), R^(2B), R^(2C),R^(3A), R^(3B), R^(3C), R^(4A), R^(4B), R^(4C), R^(5A), R^(5B), R^(5C),R^(6A), R^(6B), R^(6C), R^(7A), R^(7B), R^(7C) or a combination thereof.Alternatively, as shown here, the monomer described herein can beintegrated with the polymer by attachment as shown by the parentheses.

Hybrid polymer dots of the present disclosure can include a polymer thatincludes a narrow-band monomer having the structure of Formula (XXVII):

wherein each of R^(1A) and R^(1B) is independently selected from thegroup consisting of, but not limited to, hydrogen, deuterium, alkyl andaryl; and each of R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R⁵,R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independently selected from thegroup consisting of, but not limited to, hydrogen, deuterium, alkyl,aryl, cyano, amino, sulfide, aldehyde, ester, ether, carboxyl, hydroxyl,and halide. The narrow-band monomer can be integrated into a backbone ofthe polymer (e.g., along the backbone of the polymer by polymerizing inthe polymer) and/or attached by covalent attachment to the backbone, aterminus, or a sidechain of the polymer through at least one attachmentto R^(1A), R^(1B), R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R⁵,R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² or a combination thereof.

Hybrid polymer dots of the present disclosure can include a polymer thatincludes a narrow-band monomer having the structure of Formula (XXVIII):

wherein each of R^(13A), R^(13B) is independently selected from thegroup consisting of, but not limited to, hydrogen, deuterium, alkyl andaryl; and R^(14A), R^(14B), R^(15A), R^(15B), R^(16A), R^(16B), R¹⁷,R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³ and R²⁴ are each independently selectedfrom the group consisting of, but not limited to, hydrogen, deuterium,alkyl, aryl, cyano, amino, sulfide, aldehyde, ester, ether, carboxyl,hydroxyl, and halide. The narrow-band monomer can be integrated into abackbone of the polymer (e.g., along the backbone of the polymer bypolymerizing in the polymer) and/or attached by covalent attachment tothe backbone, a terminus, or a sidechain of the polymer through at leastone attachment to R^(13A), R^(13B), R^(14A), R^(14B), R^(15A), R^(15B),R^(16A), R^(16B), R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³ and R²⁴ or acombination thereof.

In some embodiments, the narrow-band emissive polymers for making hybridpolymer dots include metal complexes and their derivatives asnarrow-band monomers. Metal complexes and their derivatives include butare not limited to their alkyl derivatives, aryl derivatives, alkynederivatives, aromatic derivatives, alkoxide derivatives, azaderivatives, their extended systems and analogues. The narrow-bandemissive polymers can also include any other monomers. The metals can beany metal such as Na, Li, Zn, Mg, Fe, Mn, Co, Ni, Cu, In, Si, Ga, Al,Pt, Pd, Ru, Rh, Re, Os, Ir, Ag, Au and so on. In some embodiments themetal can be Fe, Ni, Co, Ga, or Au. In some embodiments the metal can beFe, Ni, Co, Ga, oxides thereof, alloys thereof, complexes thereof,combinations thereof, and combinations and complexes with non-magneticor non-magnetic metals. The metal complexes can be energy acceptors andother monomers can be energy donors so that the final hybrid polymerdots can exhibit narrow-band emissions. The narrow-band emissivechromophoric polymers in good solvents may exhibit broad emissions ornarrow emissions. However, in some embodiments, their nanoparticle formgives narrow-band emissions. In some embodiments, the emission FWHM ofthe above hybrid polymer dots is less than 70 nm. In certainembodiments, the FWHM is less than 60 nm, less than 50 nm, less than 40nm, less than 30 nm, or less than 20 nm. Metal complexes and metalcomplex derivatives can be polymerized to form polymers (e.g.,homopolymers or heteropolymers) and/or can be attached (e.g., covalentlyattached) to a polymer backbone, sidechain and/or terminus.

In some embodiments, the narrow-band emissive polymers for making hybridpolymer dots include porphyrin, metalloporphyrin, and their derivativesas narrow-band monomers. Porphyrin, metalloporphyrin, and theirderivatives can be polymerized to form polymers (e.g., homopolymers orheteropolymers) and/or can be attached (e.g., covalently attached) to apolymer backbone, sidechain and/or terminus. Porphyrin,metalloporphyrin, and their derivatives include but are not limited totheir alkyl derivatives, aryl derivatives, alkyne derivatives, aromaticderivatives, alkoxide derivatives, aza derivatives, their extendedsystems and analogues. The metals in the metalloporphyrins can be anymetal such as Na, Li, Zn, Mg, Fe, Mn, Co, Ni, Cu, In, Si, Ga, Al, Pt,Pd, Ru, Rh, Re, Os, Ir, Ag, Au and so on. In some embodiments the metalcan be Fe, Ni, Co, Ga, or Au. In some embodiments the metal can be Fe,Ni, Co, Ga, oxides thereof, alloys thereof, complexes thereof,combinations thereof, and combinations and complexes with magnetic ornon-magnetic metals. The narrow-band emissive polymers can also includeany other monomers. The porphyrin, metalloporphyrin and theirderivatives can be energy acceptors and other monomers can be energydonors so that the final hybrid polymer dots can exhibit narrow-bandemissions. The narrow-band emissive chromophoric polymers in goodsolvents may exhibit broad emissions or narrow emissions. However, insome embodiments, their nanoparticle form gives narrow-band emissions.In some embodiments, the emission FWHM of the above hybrid polymer dotsis less than 70 nm. In certain embodiments, the FWHM is less than 60 nm,less than 50 nm, less than 40 nm, less than 30 nm, or less than 20 nm.

In some embodiments, the narrow-band emissive polymers for making hybridpolymer dots include phthalocyanine and its derivatives as monomers.Phthalocyanine and its derivatives as monomers can be polymerized toform polymers (e.g., homopolymers or heteropolymers) and/or can beattached (e.g., covalently attached) to a polymer backbone, sidechainand/or terminus. Phthalocyanine derivatives include but are not limitedto their alkyl derivatives, aryl derivatives, alkyne derivatives,aromatic derivatives, alkoxide derivatives, aza derivatives, theirextended systems and analogues. The metals in the phthalocyaninederivatives can be any metal such as Na, Li, Zn, Mg, Fe, Mn, Co, Ni, Cu,In, Si, Ga, Al, Pt, Ru, Rh, Re, Os, Ir, Ag, Au or Pd. In someembodiments the metal can be Fe, Ni, Co, Ga, or Au. In some embodimentsthe metal can be Fe, Ni, Co, Ga, oxides thereof, alloys thereof,complexes thereof, combinations thereof, and combinations and complexeswith magnetic or non-magnetic metals. The narrow-band emissive polymerscan also include any other monomers. The phthalocyanine derivatives canbe energy acceptors so that the final hybrid polymer dots can exhibitnarrow-band emissions. The narrow-band emissive chromophoric polymers ingood solvents may exhibit broad emissions or narrow emissions. However,in some embodiments, their nanoparticle form gives narrow-bandemissions. In some embodiments, the emission FWHM of the above hybridpolymer dots is less than 70 nm. In certain embodiments, the FWHM isless than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, orless than 20 nm.

A wide variety of chromophoric polymer particles can be used inaccordance with the embodiments herein, such as the examples describedherein as well as others that are disclosed, e.g., in PCT/US2010/056079and PCT/US2012/071767, each of which is incorporated by reference hereinit its entirety and specifically with regard to the particularchromophoric polymer particle compositions and the respective methods ofmaking them as described therein.

Functionalization and Bioconjugation of Hybrid Polymer Dots

In some embodiments, the present disclosure provides hybrid polymer dotshaving a functional group (e.g., “X”) that is physically and/orchemically attached to the polymer dot, also referred to herein as afunctionalized hybrid polymer dot. In some embodiments, the term“functional group” refers to any chemical unit that can be attached,such as by any stable physical or chemical association, to the hybridpolymer dot, thereby rendering the surface of the hybrid polymer dotavailable for conjugation or bioconjugation. In some embodiments,functionalization occurs such that functional groups suitable forbioconjugation are oriented on the surface of the polymer dot. Forexample, the organic-inorganic hybrid polymer dots herein can have asurface that is functionalized with a functional silane speciescomprising a carboxyl, an amine, a thiol (—SH), a carboxylate orcarboxylic acid, a maleimide, a maleic anhydride, a N-hydroxysuccinimide(NHS), an alcohol (—OH), or a cyanate, or a combination thereof, that issuitable for bioconjugation.

In some embodiments, functional groups can be hydrophobic functionalgroups. Examples of hydrophobic functional groups include but notlimited to alkyne, strained alkyne, azide, diene, alkene, cyclooctyne,and phosphine groups (for click chemistry). In some embodiments,functional groups can be hydrophilic functional groups. Examples ofhydrophilic functional groups include but not limited to carboxylic acidor salts thereof, amino, mercapto, azido, diazo, aldehyde, ester,hydroxyl, carbonyl, sulfate, sulfonate, phosphate, cyanate, succinimidylester, substituted derivatives thereof. Such functional groups can befound by one of ordinary skill in the art, for example in BioconjugateTechniques (Academic Press, New York, 1996 or later versions) thecontent of which is herein incorporated by reference in its entirety forall purposes.

In some embodiments, hybrid polymer dots are functionalized usingfunctional groups including, without limitation, any the following: analdehyde, alkene, alkyl, alkyne, strained alkyne, amino, azido,carbonyl, carboxyl, cyano, cyclooctyne, dieno, ester, succinimidylester, haloalkyl, hydroxyl, imido, ketone, maleimido, mercapto,phosphate, phosphine, sulfate, sulfonate, substituted derivativesthereof, or combination thereof.

In some embodiments, a functional group is created with covalent bondingto the backbone, side chain, or terminating unit of the chromophoricpolymer. Therefore, the resulting hybrid polymer dots exhibitnarrow-band emission and simultaneously have functional groups forbioconjugation. Such functional groups could be found by one of ordinaryskill in the art, for example in Bioconjugate Techniques (AcademicPress, New York, 1996 or later versions) the content of which is hereinincorporated by reference in its entirety for all purposes. In someembodiments, each hybrid polymer dot has only one functional group. Insome embodiments, each hybrid polymer dot has only two functionalgroups. The two functional groups can be the same or different. In someembodiments, each hybrid polymer dot has three or more functionalgroups. The three or more functional groups can be the same ordifferent.

In certain embodiments of the present disclosure, the degree offunctionalization of the hybrid polymer dot can be varied as desired. Insome embodiments, the hybrid polymer dots provided herein are modifiedto form a single-molecule polymer particle that can be monovalent,bivalent, or multivalent. The modification is to remove some polymermolecules from the particle, but leave only one molecule that can havejust one functional group, two or more functional groups. In oneembodiment, an engineered surface can be used to facilitate themodification. The engineered surface can have certain functional groupssuch as aldehyde, alkene, alkyl, alkyne, strained alkyne, amino, azido,carbonyl, carboxyl, cyano, cyclooctyne, dieno, ester, succinimidylester, haloalkyl, hydroxyl, imido, ketone, maleimido, mercapto,phosphate, phosphine, sulfate, sulfonate, substituted derivativesthereof, and combinations thereof. In general, any other functionalgroups that are suitable for bioconjugation can be used. Such functionalgroups could be found by one of ordinary skill in the art, for examplein Bioconjugate Techniques (Academic Press, New York, 1996 or laterversions). The surface can be a flat surface such as a coverslip or acurved surface from any particles. The surfaces can be silica, metal,semiconducting, silicon, and different polymer surfaces. Thefunctionalized multi-molecule hybrid polymer dot described above isattached to the surface by only one chromophoric polymer molecule viaany stable physical or chemical association. All the free molecules(except the one associated with the surface) in the hybrid polymer dotcan be removed, such as by washing the surface with an organic solvent,so that only the molecule associated with the surface is retained. Thenthe single-molecule hybrid polymer dot can be released from the surfaceby any physical or chemical methods. The resulting single-moleculeparticle could be monovalent, bivalent, or multivalent, depending on thenumber of functional groups in the original polymer molecule. In anotherembodiment, all the functional groups (except the one associated withthe surface) in the hybrid polymer dot can be inactivated or reacted toform other types of functional groups or non-reactive chemical groupsfor bioconjugation, such that after release from the surface, theremaining functional group (the one attached to the surface) can be usedfor bioconjugation.

In some embodiments, advantages can arise from using hybrid polymer dotsthat include a single polymer molecule having at least one functionalgroup at a terminal unit. For example, the attachment of only onefunctional group to a terminal unit of a chromophoric polymer can bewell controlled in polymer synthesis. For example, a chemical unitcomprising a functional group can serve as a polymerization initiator aswell as a growth catalyst in polymer synthesis, and in this way eachpolymer molecule includes just one functional group at the terminus.Attachment of functional groups only to the two terminal units of alinear chromophoric polymer can also be well controlled in polymersynthesis. For example, a chemical unit comprising a functional groupcan be used as a capping agent to terminate the polymer growth inpolymer synthesis, thereby resulting in each linear polymer moleculeincluding only two functional groups in the two terminal units.Similarly, the attachment of functional groups for multivalent polymerparticles can be well controlled in polymer synthesis, e.g., functionalgroups can only be added to the three terminal units of a three-armbranched polymer.

In some embodiments, the present disclosure provides a bioconjugate ofthe hybrid polymer dot. The bioconjugates also include hybrid polymerdots as described above associated with biological particles such asviruses, bacteria, cells, biological or synthetic vesicles such asliposomes, or combinations thereof. In some embodiments, the terms“biomolecule” or “biological molecule” are used interchangeably todescribe a synthetic or naturally occurring protein, glycoprotein,peptide, amino acid, metabolite, drug, toxin, nuclear acid, nucleotide,carbohydrate, sugar, lipid, fatty acid, and the like, or combinationsthereof. In some embodiments, the biomolecule is a polypeptide or apolynucleotide. In some embodiments, the biomolecule is an antibody, anavidin, a biotin, a nucleic acid, or a combination thereof. In someembodiments, the bioconjugate is formed by the attachment of abiomolecule to one or more functional groups of the hybrid polymer dot.The attachment may be direct or indirect. Optionally, the biomolecule isattached to the functional group of the hybrid polymer dot via acovalent bond. For example, if the functional group of the polymerparticle is a carboxyl group, a protein biomolecule can be directlyattached to the hybrid polymer dot by cross-linking the carboxyl groupwith an amine group of the protein molecule. In some embodiments, eachpolymer particle has only one type of biomolecule attached. In someembodiments, the biomolecular conjugation does not change substantivelythe emissive properties of the hybrid polymer dot. For example, thebioconjugation does not substantively change the emission spectra, doesnot reduce fluorescence or luminescence quantum yield, does notsubstantively change the photostability, etc.

Some of the functional groups of a hybrid polymer dot can be “suitablefor bioconjugation,” which refers to a functional group that is or thatis capable of being covalently bonded to a biomolecule, such as anantibody, protein, nucleic acid, streptavidin, or other molecule ofbiological relevance. Functional groups can render the surface of thehybrid polymer dots available for conjugation or bioconjugation. Thehybrid polymer dots can include one or more functional groups that areformed from the siloxane network. Such functional groups can be found byone of ordinary skill in the art, for example in Bioconjugate Techniques(Academic Press, New York, 1996 or later versions) the content of whichis herein incorporated by reference in its entirety for all purposes. Insome embodiments, functional groups suitable for bioconjugation caninclude functional groups that can be conjugated to a biomolecule undera variety of conditions, such as, e.g., in polar or non-polar solvents.In certain embodiments, functional groups suitable for bioconjugationcan include functional groups that can be conjugated to a biomolecule inan aqueous solution. In some embodiments, functional groups suitable forbioconjugation can include functional groups that can be conjugated to abiomolecule in an aqueous solution in which the biomolecule retains itsbiological activity (e.g., monoclonal binding specificity for anantibody).

In certain embodiments, functional groups suitable for bioconjugationare covalently bonded to a biomolecule. For example, typical covalentbonding attachments of functional groups to biomolecules can include,e.g., a carboxyl functional group reacting with an amine on abiomolecule to form an amide bond, a sulfhydryl functional groupreacting with a sulfhydryl group on a biomolecule to form a cysteinebond, or an amino functional group reacting with a carboxyl group on abiomolecule to form an amide bond. A biomolecule can be attached to ahybrid polymer dot either directly or indirectly by the functionalgroups so as to form a bioconjugate. The biomolecule can be attached toa functional group of a hybrid polymer dot via a covalent bond. Forexample, if the functional group of the hybrid polymer dot is a carboxylgroup, a protein biomolecule can be directly attached to the polymer dotby cross-linking the carboxyl group with an amine group of the proteinmolecule.

Functional groups suitable for bioconjugation can comprise an amine, acarboxylate, a carboxyl, a maleimide, a thiol (—SH), a maleic anhydride,an N-hydroxysuccinimide ester, a mercapto, an azido, an alkyne, analdehyde, a hydroxyl, a carbonyl, a sulfate, a sulfonate, a phosphate, acyanate, a succinimidyl ester, a strained alkyne, an azide, a diene, analkene, a tetrazine, a strained alkene, a cyclooctyne, or a phosphine.In some embodiments, a functional group suitable for bioconjugation is acarboxyl group.

Indirect attachment of the biomolecule to hybrid polymer dots can occurthrough the use of a linker moiety (e.g., “L”), for example, avidin,streptavidin, neutravidin, biotin, or the like. Linker moieties can beselected from a chemical bond, an amino acid, an ester, an amide, acarbamate, an ether, an alkylene, an alkenylene, an alkynylene, anarylene, a poly ether, a polyester, a polyamide, a poly carbamate, apolyaryl, a polystyrene, polyethylene glycol, or a polyolefin, or afluorinated or partially fluorinated derivative thereof, or acombination thereof. In some embodiments, a linker moiety isamphiphilic. In some embodiments, a linker moiety is a water-solublepolymer. For example, the water-soluble polymer can be polyethyleneglycol. In some embodiments, a linker moiety is a chemical bond.

A functional group suitable for bioconjugation can be combined with alinker moiety to facilitate bioconjugation. In the hybrid polymer dotsdescribed herein, at least one functional group suitable forbioconjugation combined with a linker moiety (e.g., “D” or “LX”) can bepositioned on the surface of the hybrid polymer dot. In someembodiments, a biological molecule is conjugated to functional groupsuitable for bioconjugation combined with a linker moiety. In someembodiments, a biological molecule is conjugated to a functional groupsuitable for bioconjugation combined with a linker moiety positioned onthe surface of the hybrid polymer dot. In some embodiments, thebiological molecule comprises a protein or a nucleic acid. In someembodiments, the biological molecule comprises an antibody. In someembodiments, the biological molecule comprises streptavidin.

In various embodiments of the present disclosure cross-linking agentscan be utilized to facilitate bioconjugation of hybrid polymer dots. Insome embodiments, the term “cross-linking agent” is used to describe acompound or moiety that is capable of forming a chemical bond betweenmolecular groups on similar or dissimilar molecules so as to covalentlybond together the molecules. Examples of common cross-linking agents areknown in the art. See, for example, Bioconjugate Techniques (AcademicPress, New York, 1996 or later versions). Indirect attachment of thebiomolecule to hybrid polymer dots can occur through the use of “linker”molecules, for example, avidin, streptavidin, neutravidin, biotin or alike molecule.

In some embodiments, analysis of a target analyte molecule (e.g., aprotein) is achieved using hybrid polymer dots conjugated tobiomolecules that specifically bind to the target analyte.

In some embodiments, fluorescent and/or luminescent hybrid polymer dotsare conjugated to one or more molecules that provide a function or otherbenefit, including without limitation, binding affinity for a targetanalyte.

In some embodiments, the analyte is a polypeptide, a polynucleotide, acell, a virus, a small molecule, a drug, a toxin, a carbohydrate, asugar, a lipid, or a fatty acid.

In some embodiments, the target analyte molecule is a polypeptide, suchas a protein, and the biomolecule conjugated to a hybrid polymer dot isa primary antibody that specifically binds to the target analyteprotein.

In other embodiments, the target analyte molecule is a protein ofinterest bound to a primary antibody for said protein, and thebiomolecule conjugated to a hybrid polymer dot is a secondary antibodythat specifically binds to the primary antibody.

In other embodiments, the target analyte molecule is a biotinylatedprotein of interest, and the biomolecule conjugated to a hybrid polymerdot is an avidin (e.g., streptavidin) that specifically binds to thebiotinylated protein.

In some embodiments, the term “biotin” refers to any one of a variety ofbiotin derivatives and analogs that are effective in avidin binding.Suitable biotin moieties include those moieties that enable thebiotinylated peptide fragment to be isolated by avidin and relatedavidin proteins. Representative biotin moieties include biotinderivatives such as iminobiotin, biocytin, and caproylamidobiotin, andbiotin analogs such as desthiobiotin and biotin sulfone.

In some embodiments, the term “avidin” refers to any biotin-bindingprotein other than an immunoglobulin that binds biotin including bothnatural proteins and recombinant and genetically engineered proteins.The term includes the two common biotin-binding proteins known as “eggwhite” or “avian” avidin and “streptavidin.” Egg white or avian avidin,commonly referred to simply as avidin, is a protein that is aconstituent of egg white and forms a noncovalent complex with biotin.Streptavidin is an avidin protein isolated from the actinobacteriumStreptomyces avidinii and also forms a noncovalent complex with biotin.Other bacterial sources of biotin binding proteins are also known. Bothegg white avidin and streptavidin are tetrameric proteins in which thebiotin binding sites are arranged in pairs on opposite faces of theavidin molecule. The term also refers to avidin derivatives includingsuccinyl avidin, ferritin avidin, enzyme avidin, and crosslinked avidin.

In some embodiments, the target analyte molecule is a polynucleotide,such as DNA, RNA, or PNA, and the biomolecule conjugated to a hybridpolymer dot is a complementary polynucleotide that specifically binds tothe target analyte polynucleotide.

In some embodiments, hybrid polymer dots may be conjugated to one ormore molecules that alter other properties of the polymer particles,such as their size, fluorescence, hydrophobicity, non-specific bindingor adsorption properties, and the like.

In some embodiments, conjugation of biomolecules to hybrid polymer dotscan include attachment of a functional group, including but not limitedto attachment of carboxyl groups to polymer particles. In someembodiments, carboxyl groups can be reacted to N-hydroxysuccinimide(NHS) in the presence of a carbodiimide such asl-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) toproduce amine-reactive esters of carboxylate groups for crosslinkingwith primary amine groups present on certain biomolecules.

In some embodiments, carboxylated hybrid polymer dots are conjugated toa biomolecule, such as a protein, by mixing of the hybrid polymer dotsand the biomolecules, e.g., in a HEPES buffer (20 mM, pH=7.4) solutioncontaining 0.1 PEG (MW3350). Formation of a peptide bond between thecarboxyl groups on polymer particles and the amine groups of thebiomolecule can be catalyzed by EDC. However, in some embodiments, dueto the intrinsically hydrophobic nature of the polymer particles,biomolecules tend to nonspecifically adsorb onto the particle surface.In some embodiments, Triton X-100 and/or bovine serum albumin (BSA) areintroduced to reduce non-specific adsorption of a biomolecule onto thesurface of a polymer particle.

In addition to the examples described herein, in some embodiments otherstrategies and methods for conjugation of biomolecules to hybrid polymerdots can be used, including those disclosed, e.g., in PCT/US2010/056079and PCT/US2012/071767. Other strategies and methods for conjugation ofbiomolecules to hybrid polymer dots can be found by one of ordinaryskill in the art, for example in Bioconjugate Techniques (AcademicPress, New York, 1996 or later versions).

Methods of Making Organic-Inorganic Hybrid Polymer Dots

The present disclosure provides methods of making hybrid polymer dots asdisclosed herein. In certain embodiments, one or more polymers arecollapsed, precipitated, or condensed to form an organic network and aninorganic network, which together form the organic-inorganicinterpenetrated network as described herein.

For example, the methods can comprise: providing a solution (e.g., anorganic solution), wherein the solution comprises a solvent, asemiconducting chromophoric polymer, and an organo-silane; and mixingthe solution with an aqueous solution, wherein the solution, the aqueoussolution, or both comprise an organo-silane comprising X, where X is afunctional group suitable for bioconjugation. For instance, if theorgano-silane comprising X is relatively hydrophobic, it can be providedin the solution with the solvent. Conversely, if the organo-silanecomprising X is relatively hydrophilic, it can be provided in theaqueous solution.

FIG. 1 shows an exemplary schematic illustration for preparing hybridPdots. In some embodiments, an organic solution (e.g., a THF solution)including a polymer (e.g., chromophoric semiconducting polymer) and analkyl silane is provided. The organic solution is injected into anaqueous solution. In certain embodiments, the organic solution includesa silane with one or more functional groups (e.g., X). Alternatively orin combination, the aqueous solution includes a silane with one or morefunctional groups (e.g., X). Introduction of the organic solution intothe aqueous solution produces hybrid polymer dots with functionalgroups.

Optionally the organic solution further includes a silane. In someembodiments, inclusion of a silane in the organic solution reduces theoverall size of the hybrid resultant polymer dot.

In some embodiments, the organo-silane is selected from:

wherein: A¹, A², A³, A⁶, A⁷, A¹¹, A¹⁶, A¹⁷, A¹⁸, A¹⁹, A²⁰, and A²¹ areeach independently C_(n)H_(2n+1), C_(n)H_(2n)X, C_(n)F_(2n+1), orC_(n)F_(2n)X; A⁰, A⁴, A⁵, A⁸, A⁹, A¹⁰, A¹², A¹³, A¹⁴, and A¹⁵ are eachindependently C_(m)F_(2m+1), C_(m)H_(2m)X, C_(m)F_(2m+1), orC_(m)F_(2m)X; n is not less than 1; and m is not less than 1. In someembodiments, n is not less than 2, not less than 3, not less than 4, notless than 5, not less than 6, not less than 7, not less than 8, not lessthan 9, or not less than 10. In some embodiments, n is not less than 6.In some embodiments, n is not greater than 20. In some embodiments, n isnot greater than 40. In some embodiments, n is not greater than 60. Insome embodiments, n is not less than 6 and is not greater than 20. Insome embodiments, n is not less than 6 and is not greater than 15. Insome embodiments, n is not less than 6 and is not greater than 10. Insome embodiments, m is not less than 2, not less than 3, not less than4, not less than 5, not less than 6, not less than 7, not less than 8,not less than 9, or not less than 10. In some embodiments, m is notgreater than 5, not greater than 6, not greater 7, not greater than 8,not greater than 9, or not greater than 10. In some embodiments, m isnot greater than 20. In some embodiments, m is not greater than 40. Insome embodiments, m is not greater than 60. In some embodiments, m isnot less than 1 and is not greater than 20.

The organo-silane comprising X can be selected from:

wherein: A⁰, A¹, A², A³, A⁴, and A⁵, are each independentlyC_(m)H_(2m+1), C_(m)H_(2m)X, C_(m)F_(2m+1), or C_(m)F_(2m)X; D is LX,wherein L is a linker moiety; and m is not less than 1. In someembodiments, L can be, but is not limited to, an amino acid, an ester,an amide, a carbamate, an ether, an alkylene, an alkenylene, analkynylene, an arylene, a polyether, a polyester, a polyamide, a polycarbamate, a polyaryl, a polystyrene, or a polyolefin, or a fluorinatedor partially fluorinated derivative thereof, or a combination thereof.In some embodiments, L is a water-soluble polymer. In certainembodiments, the water-soluble polymer can be polyethylene glycol. Inother embodiments, L can be a chemical bond. In some embodiments, m isnot less than 3. In some embodiments, m is not less than 6. In someembodiments, m is not greater than 5, not greater than 6, not greater 7,not greater than 8, not greater than 9, or not greater than 10. In someembodiments, m is not greater than 20. In some embodiments, m is notgreater than 40. In some embodiments, m is not greater than 60. In someembodiments, m is not less than 1 and is not greater than 20.

In some embodiments, n is not less than 1, not less than 2, not lessthan 3, not less than 4, not less than 5, not less than 6, not less than7, not less than 8, not less than 9, or not less than 10. In someembodiments, n is not less than 6. In some embodiments, n is not greaterthan 20. In some embodiments, n is not greater than 40. In someembodiments, n is not greater than 60. In some embodiments, n is notless than 6 and is not greater than 20.

In some embodiments, m is not less than 1, not less than 2, not lessthan 3, not less than 4, not less than 5, not less than 6, not less than7, not less than 8, not less than 9, or not less than 10. In someembodiments, m is not less than 6. In some embodiments, m is not greaterthan 20. In some embodiments, m is not greater than 40. In someembodiments, m is not greater than 60. In some embodiments, m is notless than 1 and is not greater than 20.

The organo-silane can be selected from an alkyl silane, an alkoxysilane, a chloro silane, an orthosilicate, a siloxane, an alpha silane,an acetoxy silane, an amino silane, a bis silane, an epoxy silane, ahalo silane, a hydrogen silane, a hydroxyl silane, an ester silane, anaryl silane, an acryl silane, a methacryl silane, a styryl silane, avinyl silane, an olefin silane, a sulfur silane, a phosphine silane, aphosphate silane, an isocyanate silane, an azide silane, an anhydridesilane, or a hydrogen siloxane, or a combination thereof. Specifically,the organo-silane can be selected from octodecyltrimethoxysilane,octodecyltrichlorosilane, tetraethylorthosilicate,trifluoropropyltrimethoxysilane, phenyltrimethoxysilane,chloropropyltrimethoxysilane, heptadecafluorodecyltrichlorosilane,glycidoxypropyltrimethoxysilane, epoxyhexyltriethoxysilane,hydroxymethyltriethoxysilane, iodopropyltrimethoxysilane,isocyantopropyltrimethoxysilane, methacryloxymethyltriethoxysilane,vinyltrimethoxysilane, styrylethyltrimethoxysilane, or a combinationthereof. More specifically, the organo-silane can be selected fromoctodecyltrimethoxysilane, octodecyltrichlorosilane, ortetraethylorthosilicate, or a combination thereof.

In some embodiments, an organo-silane is used to form a hybrid polymerdot including a siloxane network, and the weight percent of the siloxanenetwork and/or the components thereof (e.g., silicon) in a hybridpolymer dot can be varied as desired. In some embodiments, the weightpercent of the siloxane network and/or the components thereof (e.g.,silicon) is selected to avoid formation of a core-shell structure in theresulting hybrid polymer dot. In certain embodiments, the weight percentof silicon from the siloxane network in the hybrid polymer dot is lessthan or equal to about 1%, less than or equal to about 5%, less than orequal to about 10%, less than or equal to about 15%, less than or equalto about 20%, less than or equal to about 25%, less than or equal toabout 30%, less than or equal to about 35%, less than or equal to about40%, less than or equal to about 45%, or less than or equal to about47%. In certain embodiments, the weight percent of silicon from thesiloxane network in the hybrid polymer dot is greater than or equal toabout 1%, greater than or equal to about 5%, greater than or equal toabout 10%, greater than or equal to about 15%, greater than or equal toabout 20%, greater than or equal to about 25%, greater than or equal toabout 30%, greater than or equal to about 35%, greater than or equal toabout 40%, or greater than or equal to about 45%. In certainembodiments, the weight percent of silicon from the siloxane network inthe hybrid polymer dot is within a range from about 1% to about 45%, orwithin a range from about 1% to about 47%.

In some embodiments, the methods comprise providing a firstorgano-silane including a functional group and, optionally, a secondorgano-silane. The first organo-silane can be provided in an aqueoussolution, in an organic solution, or both. The optional secondorgano-silane can be provided in an organic solution. The organic andaqueous solutions can be combined in order to form hybrid polymer dots.

In some embodiments, the methods disclosed herein can comprise heatingthe organic solution or the aqueous solution, or a combination thereof.The aqueous solution can be alkaline. In some embodiments, the aqueoussolution can have a pH not less than 9. In some embodiments, the aqueoussolution can have a pH of not less than 10 and not greater than 11.

The aqueous solution can be acidic. In some embodiments, the aqueoussolution has a pH of not greater than 6. In some embodiments, theaqueous solution has a pH of not greater than 5. In some embodiments,the aqueous solution has a pH of not greater than 4.

In some embodiments, the hybrid polymer dots made according to themethods disclosed herein can comprise a plurality of polymers, such asone or more of the chromophoric polymers described herein. In someembodiments, the polymer dot can comprise a plurality of semiconductingchromophoric polymers. In some embodiments, the polymer dot comprises ablend of semiconducting polymers. In some embodiments, the polymer dotcan comprise a blend of semiconducting polymers and non-semiconductingpolymers. In some embodiments, the polymer dot can comprisesemiconducting chromophoric polymer. In some embodiments, the polymerdot can comprise a blend of semiconducting chromophoric polymers. Insome embodiments the semiconducting chromophoric polymer can comprise afluorene polymer, a fluorene-based polymer or copolymer, a phenylenevinylene-based polymer or copolymer, a phenylene ethynylene-basedpolymer or copolymer, or a BODIPY-based polymer or copolymer. In otherembodiments, the semiconducting chromophoric polymer can comprisepoly(9,9-dihexylfluorenyl-2,7-diyl) (PDHF),Poly(9,9-dioctylfluorenyl-2,7-diyl) (PFO),poly[{9,9-dioctyl-2,7-divinylene-fluorenylene}-alt-co-{2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylene}](PFPV),poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1,4-benzo-{2,1,3}-thiadiazole)](PFBT),poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,7-Di-2-thienyl-2,1,3-benzothiadiazole)](PFTBT),poly[(9,9-dioctylfluorenyl-2,7-diyl)-9-co-(4,7-Di-2-thienyl-2,1,3-benzothiadiazole)](PF-0.1TBT)), poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene](MEH-PPV), poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-(l-cyanovinylene-1,4-phenylene)](CN-PPV), or a semiconducting polymer comprising BODIPY monomer andemitting units, including BODIPY 570, BODIPY 590, or BODIPY 690. Thepolymer dot can comprise a BODIPY derivative. In some embodiments, theBODIPY derivative has the structure of Formula (I):

wherein each of R¹, R^(2A), R^(2B), R^(3A), R^(4A) and R^(4B) isindependently selected from hydrogen, alkyl, aralkyl, aryl, andalkoxy-aryl, and wherein the BODIPY derivative is integrated into thechromophoric polymer by attachment to R¹, R^(2A), R^(2B), R^(3A),R^(4A), and R^(4B), or a combination thereof.

X can comprise an amine, a carboxylate, a carboxyl, a maleimide, a thiol(—SH), a maleic anhydride, an N-hydroxysuccinimide ester, a mercapto, anazido, an alkyne, an aldehyde, a hydroxyl, a carbonyl, a sulfate, asulfonate, a phosphate, a cyanate, a succinimidyl ester, a strainedalkyne, an azide, a diene, an alkene, a tetrazine, a strained alkene, acyclooctyne, or a phosphine. Specifically, X can comprise a carboxylgroup.

L can be selected from a chemical bond, an amino acid, an ester, anamide, a carbamate, an ether, an alkylene, an alkenylene, an alkynylene,an arylene, a polyether, a polyester, a polyamide, a poly carbamate, apolyaryl, a polystyrene, or a polyolefin, or a fluorinated or partiallyfluorinated derivative thereof, or a combination thereof. Specifically,L can be a water-soluble polymer. The water-soluble polymer can bepolyethylene glycol. Alternatively, L can a chemical bond.

At least one D can be positioned on the surface of the polymer dot madeaccording to the methods disclosed herein. In some embodiments, abiological molecule is conjugated to D. In some embodiments, thebiological molecule is conjugated to at least one D positioned on thesurface of the polymer dot. In some embodiments, a biological moleculeis conjugated to a D positioned on the surface of the nanoparticle. Insome embodiments, the biological molecule can comprise a protein or anucleic acid. In some embodiments, the biological molecule can comprisean antibody. In some embodiments, the biological molecule can comprisestreptavidin.

Methods of Using Organic-Inorganic Hybrid Polymer Dots

The present disclosure further provides methods of using the hybridpolymer dots described herein. For example, the present disclosureprovides methods of fluorescence-based detection using the polymer dotsdisclosed herein as a novel class of fluorescent probe and theirbioconjugates for a variety of applications. These include but are notlimited to flow cytometry, fluorescence activated sorting,immunofluorescence, immunohistochemistry, fluorescence multiplexing, DNAand gene analysis, fluorescence in situ hybridization (FISH), polymerasechain reaction (PCR) analysis, isothermal DNA or RNA amplification basedanalysis, protein analysis, metabolite analysis, lipid analysis, Forsterresonance energy transfer (FRET)-based sensors, high throughputscreening, cell detection, bacteria detection, virus detection,biomarker detection, cellular imaging, in vivo imaging, biorthogonallabeling, Pdot sensors, Pdot transducer-based sensors,fluorescence-based biological assays such as immunoassays andenzyme-based assays, and a variety of fluorescence techniques inbiological assays and measurements. In certain embodiments, the hybridpolymer dots herein have a number of advantages for use as detectionagents, e.g., for detection of proteins or peptides such as in thecourse of Western blot analysis. Hybrid polymer dots according to thepresent disclosure can comprise any suitable polymer subunit or subunitsthat enable the detection of proteins or peptides, and in particular,proteins. Hybrid polymer dots according to the present disclosure cancomprise any suitable polymer subunit or subunits that enable thedetection of nucleic acids, and in particular, DNA or RNA.

In some embodiments, hybrid polymer dots can provide superiorphotophysical properties, such as high emission brightness forfluorescence-based detection methods. In some embodiments, hybridpolymer dots can provide superior specific-cellular-targetingcapabilities, such as minimal non-specific adsorption or interactionswith the target cell or cellular structure or immobilized biomolecules.In some embodiments, methods of fluorescence-based detection can includedetecting light emitted from an organic-inorganic hybrid polymer dotcomprising a semiconducting chromophoric polymer and an inorganicnetwork, wherein the semiconducting chromophoric polymer and theinorganic network form an organic-inorganic interpenetrated network. Theinorganic network may be, for example, a siloxane network, atitanium-oxide network, or a titanium-siloxane network, or any of theother inorganic networks described herein.

The hybrid polymer dots disclosed herein may be conjugated to biologicalmolecules, such as cells. Hybrid polymer dots can comprise chromophoricpolymers, providing a source of fluorescence which may be used to label,detect, and track such conjugated biological molecules. Such labelingmay be used, for example, for sorting of particles in flow cytometry,using methods such as fluorescence-activated cell sorting (FACS). Suchlabeling may be used, for example, for detecting the presence ofmolecules using immunoassays (e.g., ELISA). Such labeling may be used,for example, for detecting the presence of nucleic acids using nucleicacid amplification schemes, which may employ thermal cycling (e.g., PCR)or may employ isothermal schemes (e.g., LAMP, NASBA, RPA, RCA, etc.).Such labeling may be used, for example, for detecting the presence ofnucleic acids using non-amplification schemes (e.g., with MolecularBeacons). The fluorescence properties of hybrid polymer dots may bealtered via conjugation to biological particles, allowing the particlesto be sorted according to their state of conjugation. Conjugated hybridpolymer dots may also be used in optical identification of cells orother biological particles in solution, or adhered to a solid surface.Hybrid polymer dots provide the ability to label biological particleswhile remaining biocompatible and having a high density and smaller sizethan that available in many previously disclosed polymer dots.

In some embodiments, a method of detecting analytes is provided, themethod comprising contacting a sample comprising an analyte with ahybrid polymer dot. In other embodiments, the method comprisescontacting a sample comprising an analyte with a suspension of hybridpolymer dots. In some embodiments, the sample comprises blood, urine,stool, lymph, saliva, tears, or cerebrospinal fluid. In someembodiments, the sample is derived from a subject, such as a humansubject, an animal or a single-celled organism. In some embodiments, thesample comprises a living animal or tissue. In some embodiments, theanalyte has a binding affinity for a biomolecule attached to a hybridpolymer dot. In some embodiments, the analyte comprises a polypeptide, apolynucleotide, a cell, a cellular fraction, a virus, a drug, a toxin, acarbohydrate, a sugar, a lipid, or a fatty acid.

In some embodiments, the method further comprises measuring a signalemitted from the sample, the suspension, and/or a hybrid polymer dot. Insome embodiments, the method further comprises using a signal emittedfrom the sample and/or the suspension to measure the analyte.

In some embodiments, the method further comprises exciting the sample,the suspension, and/or a hybrid polymer dot with a source ofelectromagnetic radiation. In some embodiments, the electromagneticradiation passes through a spectral filter, a multichroic mirror, or acombination thereof. In some embodiments, the peak wavelength ofelectromagnetic radiation exciting the sample is between about 200 nmand about 300 nm, about 250 nm and about 350 nm, about 300 nm and about400 nm, about 350 nm and about 450 nm, about 400 nm and about 500 nm,about 450 nm and about 550 nm, about 500 nm and about 600 nm, about 550nm and about 650 nm, about 600 nm and about 700 nm, about 650 nm andabout 750 nm, about 700 nm and about 800 nm, about 750 nm and about 850nm, about 800 nm and about 900 nm, about 850 nm and about 950 nm, orabout 900 nm and about 1000 nm.

In some embodiments, the method further comprises separating the analytefrom the sample. In some embodiments, separating the analyte from thesample comprises directing a hybrid polymer dot associated with theanalyte to the flow cell of a flow cytometer or a microfluidic device.In some embodiments, separating the analyte from the sample comprisesattaching a hybrid polymer dot associated with the analyte to a solidsupport. In some embodiments, separating the analyte from the samplecomprises attaching a hybrid polymer dot associated with the analyte toa particle (e.g., bead or magnetic bead).

In some embodiments, compositions, methods and systems of the presentdisclosure are used for immunoassays including, but not limited to,immunocytochemistry, immunohistochemistry and enzyme-based assays. Insome embodiments, the immunoassay is used to detect an analytecomprising a polypeptide such as a protein. In some embodiments, anantibody is bound indirectly to a hybrid polymer dot, e.g., byconjugation to a functional group that is attached to the polymer dot.In some embodiments, the antibody is a primary antibody. In someembodiments the antibody is a secondary antibody. In some embodimentsboth a primary antibody and a secondary antibody are bound indirectly toa hybrid polymer dot. In some embodiments, the assay is performed oncells that have been dissociated from a tissue. In other embodiments,the assay is performed on intact (non-dissociated) tissue. In someembodiments, hybrid polymer dots are used to perform enzyme-basedassays, such as an enzyme-linked immunosorbent assay (ELISA).

In some embodiments, compositions, methods and systems of the presentdisclosure are used for analysis of polynucleotides, including but notlimited to polymerase chain reaction, reverse transcriptase PCR, ligasechain reaction, loop mediated amplification, reverse transcription loopmediated amplification, helicase dependent amplification, reversetranscription helicase dependent amplification, recombinase polymeraseamplification, reverse transcription recombinase polymeraseamplification, catalytic hairpin assembly reactions, hybridization chainreaction, entropy-driven catalysis, strand displacement amplification,reverse transcription strand displacement amplification, nucleic acidsequence based amplification, transcription mediated amplification,self-sustained sequence replication, single primer isothermalamplification, signal mediated amplification of RNA technology, rollingcircle amplification, hyper branched rolling circle amplification,exponential amplification reaction, smart amplification, isothermal andchimeric primer-initiated amplification of nucleic acids, multipledisplacement amplification, and/or in situ hybridization.

In some embodiments, compositions, methods and systems of the presentdisclosure are used for analysis of metabolites including lipids,sugars, nucleotides, amino acids, fatty acids and other metabolites.

In some embodiments, compositions, methods and systems of the presentdisclosure are used for detecting cells, including but not limited toeukaryotic cells in vitro, eukaryotic cells in vivo, and prokaryoticbacterial cells.

In some embodiments, compositions, methods and systems of the presentdisclosure are used for detecting organelles and other subcellularfractions including but not limited to mitochondria, endoplasmicreticulum and/or synaptosomes.

In some embodiments, compositions methods and systems of the presentdisclosure are used for detecting biomarkers in a bioassay. Thebiomarker can be, without limit, a polypeptide such as a protein, apolynucleotide such as DNA and/or RNA, a metabolite such as a lipid,fatty acid, sugar, nucleotide or amino acid, a cell, a virus or viralparticle.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

EXAMPLES

The compositions and methods of the present disclosure are furtherillustrated by the following non-limiting examples.

Example 1 Preparation of Hybrid Polymer Dots Using TEOS

This example demonstrates a method of making organic-inorganic hybridpolymer dots for subsequent characterization, bioconjugation, andbiological applications. This method includes the use of tetraethylorthosilicate (TEOS).

Three solutions of tetrahydrofuran (THF) containing a semiconductingpolymer, alkylsilane, and tetraethyl orthosilicate (TEOS), respectively,are prepared, and all of the THF solutions are mixed to form ahomogenous solution. The mixed solution is quickly injected into anaqueous solution under ultrasonication in an ultrasonic cleaning bath.The aqueous solution comprises an alkylsilane having a functional groupsuitable for bioconjugation, for example, carboxylic acid, carboxylate,or primary amine. The pH value of the aqueous solution is adjusted withammonia to a pH of approximately 11.

The hybrid polymer dots are obtained after removal of THF under heatingwith N₂ stripping. Alcohols formed during the hydrolysis of silanes andammonia are removed together with THF. The final hybrid Pdot solutionshows a pH value that is close to neutral, i.e., approximately 7.

Example 2 Preparation of Hybrid Polymer Dots without TEOS

This example demonstrates a method of making organic-inorganic hybridpolymer dots for subsequent characterization, bioconjugation, andbiological applications. This method does not include the use oftetraethyl orthosilicate (TEOS).

Two solutions of tetrahydrofuran (THF) containing a semiconductingpolymer, and alkylsilane, respectively, are prepared, and both of theTHF solutions are mixed to form a homogenous solution. The mixedsolution is quickly injected into an aqueous solution underultrasonication in an ultrasonic cleaning bath. The aqueous solutioncomprises an alkylsilane having a functional group suitable forbioconjugation, for example, carboxylic acid, carboxylate, or primaryamine. The pH value of the aqueous solution is adjusted with ammonia toa pH of approximately 11.

The hybrid polymer dots are obtained after removal of THF under heatingwith N₂ stripping. Alcohols formed during the hydrolysis of silanes andammonia are removed together with THF. The final hybrid Pdot solutionshows a pH value that is close to neutral, i.e., approximately 7.

Example 3 Preparation and Characterization of Hybrid Polymer Dots

This example demonstrates a method of making organic-inorganic hybridpolymer dots according to the method of Example 1 for subsequentcharacterization, bioconjugation, and biological applications.

Three solutions of tetrahydrofuran (THF) containing a semiconductingpolymer, alkylsilane, and TEOS, respectively, were prepared. Table 1provides the semiconducting polymers used, and additionally provides thecomposition ratios of the polymer to the alkylsilane and TEOS. Thealkylsilane used was either tetramethyl orthosilicate (TMOS) or TCOS(chemical structure shown in FIG. 2), as provided in Table 1.

All of the THF solutions were mixed so as to form a homogenous solution.The mixed solution was quickly injected into an aqueous solution underultrasonication in an ultrasonic cleaning bath. The aqueous solutioncomprises an alkylsilane with a functional group suitable forbioconjugation, for example, carboxylic acid, carboxylate, or primaryamine. The pH value of the aqueous solution is adjusted with ammonia toa pH of approximately 11.

The hybrid polymer dots were obtained after removal of THF under heatingwith N₂ stripping. Alcohols formed during the hydrolysis of silanes andammonia were removed together with THF. The final hybrid Pdot solutionshowed a pH value that is close to neutral, i.e., approximately 7.

Table 1 shows the size of the polymer dots as measured by dynamic lightscattering (DLS), the zeta potential (ξ), and the fluorescence quantumyield (QY). As shown in Table 1, the hybrid polymer dots show higherfluorescence quantum yields than those of the respective bare polymerdots. This indicates the significance of the formation oforganic-inorganic interpenetrated structures. These results alsoemphasize this general strategy for improving the optical properties andstability of the polymer dots.

TABLE 1 Size Pdots (nm) ζ (mV) QY (%) PFO Bare 21 −42 45/TOMS/TEOS(1:1:1) 24 −47 47 /TCOS/TEOS(1:1:1) 15.7 −42 51 PFPV Bare 13.5−48 7 /TMOS/TEOS(1:1:1) 13.5 −43 9.1 /TCOS/TEOS(1:1:1) 11.7 −41 11.2MEH-PPV Bare 21 −50 1.2 /TMOS/TEOS(1:1:1) 18 −41 1.7 /TCOS/TEOS(1:1:1)13.5 −39 1.8 CNPPV Bare 11.7 −42 45 /TMOS/TEOS(1:1:1) 11.7 −46 50/TCOS/TEOS(1:1:1) 8.7 −40 51 BODIPY Bare 24 −51 6 590 /TMOS/TEOS(1:1:1)24 −46 8.3 /TCOS/TEOS(1:1:1) 13.5 −45 11 PFTBT Bare 33 −45 44/TMOS/TEOS(1:1:1) 44 −50 54 /TCOS/TEOS(1:1:1) 15.7 −40 52 BODIPY Bare 16−41 19 680 /TMOS/TEOS(1:1:1) 24 −57 23 /TCOS/TEOS(1:1:1) 10 −40 26

Example 4 Preparation of Hybrid Polymer Dots using PFBT

This example demonstrates a method of making organic-inorganic hybridpolymer dots for subsequent characterization, bioconjugation, andbiological applications.

PFBT, a chromophoric polymer, was dissolved in tetrahydrofuran (THF) bystirring under inert atmosphere to make a solution with a concentrationof 1 mg/mL. TMOS, an organic alkylsilane, was dissolved in THF to make asolution with concentration of 1 mg/mL. Alternatively, TCOS can be usedas the organic alkylsilane. TEOS, an organic silane, was dissolved inTHF to make a solution with concentration of 1 mg/mL. The PFBT, TMOS (orTCOS), and TEOS solutions were diluted into THF to form 2 mL of a mixedhomogenous solution containing PFBT at a concentration of 0.1 mg/mL. A10⁻³ M aqueous solution of Silane-COONa was then prepared, and the pHvalue of this solution was adjusted to approximately 11. The 2 mLquantity of the PFBT solution mixture was quickly added to 10 mL of theabove-prepared aqueous Silane-COONa solution while sonicating themixture. THF was removed by nitrogen stripping, and the solution wasconcentrated by continuous nitrogen stripping to 2 mL on a hotplate at90° C., which was followed by filtration through a 0.2 micron filter.

FIG. 2 provides chemical structures of the chromophoric polymerpolyfluorene-benzothiadiazole, as well as organic alkylsilane moleculessuch as TMOS, TCOS, and TEOS. A functional silane molecule withcarboxylate groups, such as Silane-COONa is also illustrated in the FIG.2.

The resulting nanoparticle dispersions are clear and stable for monthswith no signs of aggregation. The hybrid polymer dots are furthercharacterized and conjugated to biomolecules for fluorescence imagingapplications.

Example 5 Preparation of Bare and Hybrid Polymer Dots

This example demonstrates a method of making organic-inorganic hybridpolymer dots according to the methods of Examples 1 or 2 for subsequentcharacterization, bioconjugation, and biological applications.

Hybrid polymer dots were prepared according to Examples 1 or 2, wherethe polymer, alkyl silane, and optional TEOS were provided as in Table2.

As shown in Table 2, the hybrid polymer dots show higher fluorescencequantum yields than those of the respective bare polymer dots (polymerdots without an inorganic network). This indicates the significance ofthe formation of organic-inorganic interpenetrated structures. Theseresults also emphasize this general strategy for improving the opticalproperties and stability of the polymer dots.

TABLE 2 Size Pdots (nm) ζ (mV) QY % PFBT bare 21 −54 15.3 PFBT/TMOS(1:1)21 −54 18.9 PFBT/TMOS(1:10) 33 −54 35.2 PFBT/TMOS/TEOS(1:1:10) 21 −4919.5 PFBT/TCOS(1:1) 15.7 −25 18.5 PFBT/TCOS(1:10) 13.5 −38 19.4PFBT/TCOS/TEOS(1:1:10) 15.7 −42 19.0

Example 6 Size, Surface Potential, and Fluorescence Quantum YieldCharacterizations of Hybrid Polymer Dots

This example demonstrates the assessment of the particle size, surfacepotential, and fluorescence quantum yield of hybrid polymer dots.

Hybrid polymer dots were prepared according to Examples 1 or 2, wherethe polymer, alkyl silane, and optional TEOS were provided as in Table3.

The particle sizes and surface potentials of the hybrid polymer dotswere measured by using Malvern Nanosizer ZS. UV-Vis absorption spectrawere recorded using a DU 720 spectrophotometer using a 1 cm quartzcuvette. Fluorescence spectra were collected with a Fluorolog-3fluorometer using a 1 cm quartz cuvette. Fluorescence quantum yields ofthe hybrid polymer dots were collected using an integrating sphere(Model C9920-02, Hamamatsu Photonics) with a 460 nm excitation from a150 W CW Xenon lamp.

Table 3 summarizes the particle size, surface potential, andfluorescence quantum yield data. As seen from Table 3, the hybridpolymer dots have comparable or smaller particles sizes as compared tothose of bare polymer dots. However, the hybrid polymer dots have higherquantum yields as compared to the bare polymer dots. This indicatesimproved fluorescence properties of the hybrid polymer dots.

TABLE 3 Size Pdots (nm) ζ (mV) QY % PFBT bare 20 ppm 28 −52.5 15.7PFBT:TMOS:TEOS 21 −38.5 17.3 20:10:10 (ppm) PFBT:TMOS:TEOS 18 −46.5 19.620:20:20 (ppm) PFBT:TCOS:TEOS 18 −46.2 16.1 20:10:10 (ppm)PFBT:TCOS:TEOS 16 −45.8 20.9 20:20:20 (PPM) PFBT:TMOS:TEOS 21 −38.8 17.620:5:5 (ppm) PFBT:TMOS:TEOS 24 −47.5 16.5 20:10:10 (ppm) PFBT:TMOS:TEOS28 −50.0 19.6 20:20:20 (ppm) PFBT:TMOS:TEOS 32 −51.2 19.9 20:40:40 (ppm)PFBT:TCOS:TEOS 18 −39.4 15.8 20:5:5 (ppm) PFBT:TCOS:TEOS 16 −51.2 16.120:10:10 (ppm) PFBT:TCOS:TEOS 18 −50.5 21.1 20:20:20 (ppm)PFBT:TCOS:TEOS 16 −38.7 17.0 20:40:40 (ppm)

Example 7 Transmission Electron Microscopy Characterization of HybridPolymer Dots

This example demonstrates the assessment of the size, morphology, andmonodispersity by transmission electron microscopy of hybrid polymerdots.

Hybrid polymer dots were prepared according to Example 1 to make hybridpolymer dots using PFBT, TMOS, and TEOS; PFBT, TCOS, and TEOS; andMEH-PPV, TMOS, and TEOS.

TEM measurements were made by placing one drop of a hybrid polymer dotdispersion on a copper grid. After evaporation of the water from thedispersion, the surface was imaged using TEM (FEI Tecnai F20, 200 kV).FIG. 3 shows representative TEM images of the hybrid polymer dots andbare polymer dots. The TEM results show that the hybrid polymer dotshave improved monodispersity as compared to the bare polymer dots.Notably, the magnified TEM images of the hybrid polymer dots show thatthe hybrid polymer dots do not have a core-shell structure or a core-capstructure. This indicates that hydrolysis of the organic silane forms asilica network, and an interpenetrated hybrid network of the silicanetwork and the semiconducting polymer is formed.

Example 8 Single-Particle Brightness of the Hybrid Polymer Dots

This example demonstrates a side-by-side single-particle emissionbrightness evaluation and comparison of hybrid polymer dots and barepolymer dots.

Hybrid polymer dots were prepared according to Example 1, where thepolymer, alkyl silane, and TEOS were provided as in Table 4.

Hybrid polymer dots were diluted in Milli-Q water, dried under vacuum oncleaned glass coverslips, and imaged on a fluorescence microscope. The488-nm laser beam from a sapphire laser (Coherent, Santa Clara, Calif.,USA) was directed into an inverted microscope (Nikon TE2000U, Melville,N.Y., USA) using lab-built steering optics. Laser excitation power wasmeasured at the nosepiece before the objective. The objective used forillumination and light collection was a 1.45 NA 100× objective (Nikon,Melville, N.Y., USA). Fluorescence signal was filtered by a 500 nm longpass filter (HQ500LP; Chroma, Rockingham, Vt., USA) and imaged on anEMCCD camera (Photometries Cascade: 512B, Tucson, Ariz. USA).Fluorescence intensity emitted per frame for a given particle wasestimated by integrating the CCD signal over the fluorescence spot.

FIG. 4 show single-particle intensity histograms obtained fromfluorescence images under identical acquisition and laser excitationconditions. The results summarized in Table 4 show that the hybridpolymer dots prepared according to the methods disclosed herein exhibitan improvement in fluorescence signal as compared to that of barepolymer dots. Fluorescence brightness is the product of the peakabsorption cross section and the fluorescence quantum yield. Based onthe quantum yield values, such an improvement indicates that theper-particle absorption cross sections of the hybrid polymer dots arecomparable to that of the bare polymer dots. This indicates that thenumber of chromophoric polymer chains packed in the hybrid polymer dotsis similar to that in the bare polymer dots. This indicates that,although bare polymer dots are generally larger than the hybrid polymerdots, the hybrid polymer dots have a more compact internal structure.Furthermore, the formation of the interpenetrated network may reduceself-quenching of the polymers when they are compacted closely together.

TABLE 4 Size Single particle brightness Pdots (nm) (CCD account) PFBTbare 21 13300 20 ppm PFBT:TMOS:TEOS 21 14300 20:10:10 (ppm)PFBT:TCOS:TEOS 21 19700 20:20:20 (ppm) PFBT:TCOS:TEOS 18 15000 20:10:10(ppm) PFBT:TCOS:TEOS 16 15700 20:20:20 (ppm)

Example 9 Biomolecular Conjugation of the Hybrid Polymer Dots for CellLabeling

This example demonstrates bioconjugation utilizing an EDC-catalyzedreaction between carboxyl groups on the hybrid polymer dots and aminegroups on biomolecules.

Hybrid polymer dots were prepared according to Example 1 to make hybridpolymer dots using PFBT, TMOS, and TEOS at ratios of 2:1:1 and 2:2:2, aswell as hybrid polymer dots using PFBT, TCOS, and TEOS at ratios of2:1:1 and 2:2:2.

60 μL of polyethylene glycol (5% w/v PEG, MW 3350) and 60 μL ofconcentrated HEPES buffer (1 M) were added to 3 mL of the hybrid polymerdot solution (50 μg/mL in MilliQ water), resulting in a hybrid polymerdot solution in 20 mM HEPES buffer with a pH of 7.3. Then, 180 μL ofstreptavidin was added to the solution and mixed well by vortexing.Alternatively, IgG antibody (1 mg/mL) can be used in place of 180 μL ofstreptavidin. 60 μL of l-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride (EDC) solution (5 mg/mL in MilliQ water) was added to thevortexed solution, and the mixture was left on a rotary shaker for 4hours at room temperature. The resulting hybrid polymer dot-streptavidinbioconjugates were separated from free biomolecules by gel filtrationusing Sephacryl HR-300 gel media.

MCF-7 and SK-BR-3 breast cancer cell lines were ordered from AmericanType Culture Collection (ATCC, Manassas, Va., USA). Cells were culturedat 37° C. in 5% CO₂ in Eagles minimum essential medium (for MCF-7) orMcCoy's 5A medium (for SK-BR-3) supplemented with 10% Fetal Bovine Serum(FBS), 50 U/mL penicillin, and 50 μg/mL streptomycin. The cells werepre-cultured prior to experiments until confluence was reached. Thecells were harvested from the culture flask by briefly rinsing withculture media, which was followed by incubation with 5 mL ofTrypsin-EDTA solution (0.25 w/v % Trypsin, 0.53 mM EDTA) at 37° C. for5-15 minutes. After complete detachment, the cells were rinsed,centrifuged, and resuspended in labeling buffer (lx PBS, 2 mM EDTA, 1%BSA). The cell concentration was determined by microscopy using ahemacytometer.

The hybrid polymer dot-streptavidin bioconjugates were used as probes todetect EpCAM. In order to label a cell-surface marker with hybridpolymer dot-streptavidin bioconjugates, live MCF-7 cells in theglass-bottomed culture dish were incubated sequentially with 5 μg/mLprimary anti-human CD326 antibody, 5 μg/mL biotinylated secondaryanti-mouse IgG (Biolegend, San Diego, Calif., USA), and 5 nM hybridPdot-streptavidin for 30 minutes each. Two washing steps were performedafter each incubation. The hybrid polymer dot-tagged cells were imagedimmediately on a fluorescence confocal microscope (Zeiss LSM 510). Asshown by the confocal imaging, the hybrid polymer dot-streptavidinbioconjugates, together with the biotinylated primary anti-EpCAMantibody, effectively labeled EpCAM on the surface of live MCF-7 cells

When the cells were incubated with hybrid polymer dot-streptavidinbioconjugates in the absence of biotin primary antibody, no fluorescencewas observed on the cell surface, which shows the highly specificbinding of the hybrid polymer dot-streptavidin bioconjugates. The lackof signal also indicated the absence of nonspecific binding in thisbiotin-streptavidin labeling system.

FIG. 6 and FIG. 8 provide fluorescence imaging of MCF cells labeled withhybrid polymer dot bioconjugates.

In addition to fluorescence imaging, flow cytometry was used to evaluatethe labeling brightness of the hybrid polymer dot-streptavidinbioconjugates. FIG. 11 shows flow cytometry results of MCF-7 cellslabeled with the hybrid polymer dots. “N−1” indicates control cellsincubated with hybrid polymer dots without streptavidin. “N−2” indicatescontrol cells incubated with hybrid polymer dot-streptavidinbioconjugates in the absence of biotinylated primary antibody. “P”indicates cells incubated with hybrid polymer dot-streptavidinbioconjugates and biotinylated primary antibody. Fluorescence wasobserved for the “P” group only, indicating the highly specific bindingof the hybrid polymer dot-streptaviding bioconjugates.

FIG. 5 shows the flow cytometry results of the MCF-7 cells labeled withhybrid polymer dot-streptavidin bioconjugates as compared to barepolymer dot bioconjugates. In FIG. 5, Pdot-1 is PFBT/PS-PEG-COOH(20%);Pdot-2 is PFBT/TMOS/TEOS=2/1/1; Pot-3 is PFBT/TMOS/TEOS=2/2/2; Pdot-4 isPFBT/TCOS/TEOS=2/1/1; Pdot-5 is PFBT/TMOS/TEOS=2/2/2; —N indicatesnegative controls, where cells were not incubated with biotinylatedprimary antibody, and were directly incubated with hybrid polymerdot-streptavidin conjugates without biotinylated primary antibody; and—P indicates positive labeling. The results indicate that the hybridpolymer dots exhibit similar or slightly higher cell-labeling brightnessas compared to the polymer dots functionalized by the PS-PEG-COOHblending method, the bare polymer dots.

Example 10 Photostability of the Hybrid Polymer Dots for Cell Labeling

This example demonstrates photostability measurements of the cellslabeled with hybrid polymer dot bioconjugates.

Hybrid polymer dots were prepared according to Example 1 to make hybridpolymer dots using PFBT, TMOS, and TEOS at ratios of 2:1:1 and 2:2:2, aswell as hybrid polymer dots using PFBT, TCOS, and TEOS at ratios of2:1:1 and 2:2:2.

MCF-7 cells were labeled as provided in Example 8. The hybrid polymerdot bioconjugate labeled cells were imaged on a fluorescence confocalmicroscope (Zeiss LSM 510). For photobleaching studies, confocalfluorescence images were recorded continuously for the cells labeledwith the hybrid polymer dots and those labeled with the polymer dotsblended with PS-PEG-COOH. Photobleaching data points were extracted byanalyzing the fluorescence images using a custom-coded Matlab program.As shown in FIG. 7 and FIG. 9, photobleaching curves extracted from thefluorescence images indicate that the hybrid polymer dot were morephotostable than the polymer dots functionalized by the PS-PEG-COOHblending method.

Example 11 Gel Electrophoresis of Hybrid Polymer Dots and RelatedBioconjugates

This example demonstrates the characterization of the functional groupson the surface of the hybrid polymer dots using gel electrophoresis.

Gel electrophoresis was performed using a 0.7% agarose gel. Agarose gelelectrophoresis of functionalized hybrid polymer dots was carried outusing a Mupid®-exU submarine electrophoresis system. The functionalizedhybrid polymer dots, in 30% glycerol, were loaded onto a 0.7% agarosegel containing 0.1% polyethylene glycol. The functionalized hybridpolymer dot-loaded gel was run for 20 min at 135 V in tris-borate-EDTA(TBE) buffer, and then imaged on a Kodak image station 440CF system. Asshown in FIG. 10, compared to unfunctionalized, bare polymer dots, thefunctionalized hybrid polymer dots exhibited an increase in mobility inthe gel. Notably, once the hybrid polymer dots are conjugated tostreptavidin, the hybrid polymer dot-streptavidin bioconjugates showdecreased mobility. This can be used to detect successfulbioconjugation.

Example 12 Determination of Network Structure for Hybrid Polymer Dots

This example demonstrates the characterization of the interpenetratednetwork generated in formation of the hybrid polymer dots utilizing TEMand flow cytometry.

Interpenetrated hybrid polymer dots were prepared as according toExample 1 using PFBT, TCOS, and TEOS, at a weight ratio of 1:1:1.

PFBT-14% C₂COOH, a functionalized chromophoric polymer, was dissolved intetrahydrofuran (THF) by stirring under inert atmosphere to make asolution with concentration of 1 mg/mL. TCOS, an organic silane, wasdissolved in THF to make a solution with concentration of 1 mg/mL. TEOSwas dissolved in THF to make a solution with concentration of 1 mg/mL.The above solutions of PFBT-14% C₂COOH, TCOS, and TEOS were diluted intoTHF to form 2 mL of a mixed homogenous solution containing PFBT-14%C₂COOH at a concentration of 0.1 mg/mL. Deionized water was obtained andthe pH value of it was adjusted to approximately 11. The 2 mL quantityof the PFBT-14% C₂COOH solution was quickly added to 10 mL of theaqueous solution while sonicating the mixture. THF was removed bynitrogen stripping, and the solution was concentrated by continuousnitrogen stripping to 2 mL on a hotplate at 90° C., which was followedby filtration through a 0.2 micron filter. This afforded hybrid polymerdots wherein the chromophoric polymer was directly functionalized withcarboxyl groups, resulting in Pdots not interpenetrated withSilane-COONa.

FIG. 15 provides chemical structures of the chromophoric polymerpolyfluorene-benzothiadiazole PFBT-14% C₂COOH, as well as organic silanemolecules such as TCOS and TEOS. A resultant polymer dot directlyfunctionalized with carboxyl is also illustrated in FIG. 15.

Hybrid polymer dot-streptavidin bioconjugates were prepared as accordingto Example 8 to make PFBT-14% C₂COOH polymer dot-streptavidinbioconjugates as well as Silane-COONa polymer dot-streptavidinbioconjugates.

Flow cytometry was used to evaluate the labeling brightness of thehybrid polymer dot-streptavidin bioconjugates, as according to Example8. FIG. 16 shows flow cytometry results of MCF-7 cells labeled with thePFBT-14% C₂COOH hybrid polymer dots or labeled with the Silane-COONahybrid polymer dots. “Negative of” indicates control cells incubatedwith hybrid polymer dot-streptavidin bioconjugates in the absence ofbiotinylated primary antibody. “Positive of” indicates cells incubatedwith the hybrid polymer dot-strepdavidin bioconjugates and biotinylatedprimary antibody. Fluorescence was observed for both of the “positive”groups, indicating the specific binding of streptavidin to carboxylfunctionality applied to both types of Pdots generated. The resultindicated that the external carboxyl availability of PFBT-14% C₂COOHhybrid polymer dots is similar to the external carboxyl availability ofSilane-COONa hybrid polymer dots. This result indicated that the shortcarboxylic acid functional group of the PFBT backbone chain inside thePdots is not encased by the silica network as a shell outside the hybridPdots, but instead exists as a part of an interpenetrated network formedbetween the polymer chains and silica network. The result of this flowcytometry experiment indicated that the hybrid polymer Pdots formed witha mesh-like structure, and do not have a distinct core-shell structure.This indicates that hydrolysis of the organic silane forms a silicanetwork which interpenetrat with the semiconducting polymers andtherefore formed a hybrid interpenetrated network.

TEM measurements were made by placing one drop of a hybrid polymer dotdispersion on a copper grid. After evaporation of the water from thedispersion, the surface was imaged using TEM (FEI Tecnai F20, 200 kV).FIG. 17 shows a representative TEM image of the PFBT-14% C₂COOH hybridpolymer dots. Notably, the magnified TEM images of the hybrid polymerdots show that the hybrid polymer dots do not have a core-shellstructure or a core-cap structure. This furthermore indicates thathydrolysis of the organic silane forms a silica network, and then insidethe hybrid Pdots an interpenetrated hybrid network between the silicanetwork and the semiconducting polymer chains is formed.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method of making an organic-inorganicinterpenetrated hybrid polymer dot, the method comprising: providing asolution, wherein the solution comprises a solvent, a semiconductingchromophoric polymer, and an organo-silane; and mixing the solution withan aqueous solution,
 2. The method claim 1, wherein at least one of thesolution or the aqueous solution comprises an organo-silane comprisingX, and wherein X is a functional group suitable for bioconjugation. 3.The method of claim 2, wherein the organo-silane is selected from:

wherein: A¹, A², A³, A⁶, A⁷, A¹¹, A¹⁶, A¹⁷, A¹⁸, A¹⁹, A²⁰, and A²¹ areeach independently C_(n)H_(2n+1), C_(n)H_(2n)X, C_(n)F_(2n+1), orC_(n)F_(2n)X; A⁰, A⁴, A⁵, A⁸, A⁹, A¹⁰, A¹², A¹³, A¹⁴, and A¹⁵ are eachindependently C_(m)H_(2m+1), C_(m)H_(2m)X, C_(m)F_(2m+1), orC_(m)F_(2m)X.
 4. The method of claim 2, wherein the organo-silanecomprising X is selected from:

wherein: A⁰, A¹, A², A³, A⁴, and A⁵, are each independentlyC_(m)H_(2m+1), C_(m)F_(2m+1), C_(m)H_(2m)X, or C_(m)F_(2m)X; D is LX,wherein L is a linker moiety; and m is not less than
 1. 5. The method ofclaim 4, further comprising a biological molecule conjugated to D. 6.The method of claim 5, wherein the biological molecule comprises aprotein or a nucleic acid.
 7. The method of claim 1, further comprisingheating the solution or the aqueous solution, or a combination thereof.8. The method of claim 1, wherein the aqueous solution is alkaline. 9.The method of claim 8, wherein the aqueous solution has a pH not lessthan 9, or wherein the aqueous solution has a pH of not less than 10 andnot greater than
 11. 10. The method of claim 1, wherein the aqueoussolution has a pH of not greater than 6; a pH of not greater than 5; ora pH of not greater than
 4. 11. An organic-inorganic interpenetratedhybrid chromophoric polymer dot comprising a semiconducting chromophoricpolymer, an inorganic network, and a functional group that is suitablefor bioconjugation.
 12. The organic-inorganic interpenetrated hybridchromophoric polymer dot of claim 11, wherein the semiconductingchromophoric polymer comprises a plurality of units, M, selected from:

wherein: R^(S) is

R^(F) is

A⁰, A⁴, A⁸, A¹², A¹⁵, A¹⁸, are each independently C_(n)H_(2n) orC_(n)F_(2n); A¹, A², A³, A⁵, A⁶, A⁷, A⁹, A¹⁰, A¹¹, A¹³, A¹⁴, A¹⁶, A¹⁷,A¹⁹ are each independently C_(m)H_(2m+1) or C_(m)F_(2m+1); X¹, X², X³,X⁴ are each independently a functional group; n is not less than 1; andm is not less than
 1. 13. The organic-inorganic interpenetrated hybridchromophoric polymer dot of claim 11, wherein the semiconductingchromophoric polymer comprises a plurality of units, M, selected from:

wherein: R^(S) is

A⁰, A⁴, A⁸, are each independently C_(n)H_(2n) or C_(n)F_(2n); A¹, A²,A³, A⁵, A⁶, A⁷, A⁹, A¹⁰, A¹¹, are each independently C_(m)H_(2m+1) orC_(m)F_(2m+1); L is a linker moiety; n is not less than 1; and m is notless than
 1. 14. The organic-inorganic interpenetrated hybridchromophoric polymer dot of claim 11, wherein the semiconductingchromophoric polymer comprises a plurality of units, M, selected from:

wherein: R^(F) is

A⁰, A³, A⁶ are each independently C_(n)H_(2n) or C_(n)F_(2n); A¹, A²,A⁴, A⁵, A⁷ are each independently C_(m)H_(2m+1) or C_(m)F_(2m+1); X¹,X², X³, X⁴ are each independently a functional group; n is not less than1; and m is not less than
 1. 15. The organic-inorganic interpenetratedhybrid chromophoric polymer dot of claim 11, wherein the inorganicnetwork comprises a siloxane network, an alumino-siloxane network, atitanium-siloxane network, a titanium oxide network, or a combinationthereof.
 16. The organic-inorganic interpenetrated hybrid chromophoricpolymer dot of claim 11, wherein the inorganic network comprises asiloxane network.
 17. The organic-inorganic interpenetrated hybridchromophoric polymer dot of claim 16, wherein the siloxane networkcomprises a plurality of interconnected units, wherein the plurality ofinterconnected units comprises a unit selected from:

wherein: A⁰, A¹, A², A³ are each independently C_(p)H_(2n+1) orC_(p)F_(2p+1); D is LX, wherein L is a linker moiety; and p is not lessthan
 1. 18. The organic-inorganic interpenetrated hybrid chromophoricpolymer dot of claim 17, further comprising a biological moleculeconjugated to D.
 19. The organic-inorganic interpenetrated hybridchromophoric polymer dot of claim 18, wherein the biological moleculecomprises a protein or a nucleic acid.
 20. The organic-inorganicinterpenetrated hybrid chromophoric polymer dot of claim 1, wherein thesemiconducting chromophoric polymer and the inorganic network form anorganic-inorganic interpenetrated network, and wherein theorganic-inorganic interpenetrated network is mesh-like.